Patent Application
20230058519
The subject matter disclosed herein relates to cells (Baize Super Cells) that express immune system modulatory proteins or other effector polypeptides, chimeric immune cell activator polypeptides (ICAPs) and to systems that are used to control the expression of such proteins and polypeptides in those cells. Such systems can include polypeptides that have bispecific binding activities and so can activate cells harboring vectors for expressing immune system modulatory proteins or other effective polypeptides upon also binding to a polypeptide target domain.
Chimeric Antigen Receptor (CAR) bearing T cells (CAR-T cells) are being developed as an immunotherapeutic mode of cancer treatment. In general, a CAR includes an extracellular domain that binds an activating ligand, a transmembrane domain that participates in forming an immune synapse with a “target” cell and an intracellular domain that responds to binding of the extracellular domain by activating T-cell associated transcriptional responses.
Present CAR-T cell based therapies are not effective against tumors with heterogeneous TAA (tumor associated antigen) expression or emerging antigen loss variants due to a single TAA recognizing extracellular domain in the CAR.
Present CAR-T cell based therapies rely on in vitro proliferation of CAR-T cells before patient treatment.
Furthermore, no easy method is available for in vivo monitoring of CAR-T cell distribution and fate.
Still further CAR-T cells continually and uncontrollably proliferate and activate in response to antigen, potentially causing fatal on-target off-tumor toxicity, cytokine release syndrome, or neurotoxicity in the absence of any a method of control of the activated CAR-T cell activity or method for elimination of undesired CAR-T cells.
Most of CAR extracellular antigen-recognizing domains are scFv proteins and it has become evident that two scFv domains can form a non-covalently linked dimer, such as by domain swapping. This type of interaction between neighboring scFv domains strongly enhances the tonic signaling in CAR-T cells, which leads uncontrollable activity.
Presently disclosed are immune cells that have been engineered to express and incorporate an immune cell activator polypeptide (ICAP) into their cell surface membrane. Also disclosed are immune cells that have been engineered to secrete one or more polypeptide effector molecules, as well as immune cells engineered to express both molecules.
Thus, in one aspect of the disclosure there is provided an immune cell that comprises a (or a first) nucleic acid vector comprising:
Alternatively, the engineered immune cell can be one wherein the first nucleic acid vector further comprises a polynucleotide encoding an amino acid sequence of one or more secreted polypeptide effector molecules.
Another aspect of the disclosure resides in an immune cell activator polypeptide comprising:
A further aspect of the disclosure is a nucleic acid vector comprising:
Another aspect of the disclosure is a nucleic acid vector comprising:
Yet another aspect of the disclosure is a bispecific polypeptide that is a nanobody targeting and control polypeptide (VHH-TCP) comprising:
The disclosure also describes a kit for in situ production of one or more polypeptide effector molecules proximal to a target cell comprising:
I. an immune cell that comprises a nucleic acid vector comprising
a second nucleic acid vector comprising
II. a bispecific polypeptide comprising:
Alternatively, the engineered immune cell can be one wherein the first nucleic acid vector further comprises a polynucleotide encoding an amino acid sequence of a secreted effector polypeptide. In such an embodiment, the secreted effector polynucleotide can be encoded in a second expression cassette. Such a kit further comprises a bispecific polypeptide that is a nanobody targeting and control polypeptide (VHH-TCP) comprising:
The disclosure also provides a method for modulating the immune system environment in the locality of a tumor cell in a subject comprising:
A step of measuring the amount of engineered immune cells in the subject can be performed between steps b and c.
A method for modulating the immune system environment in the locality of a tumor cell in a subject can alternatively comprise:
While the specification concludes with claims, which particularly point out and distinctly claim the subject matter described herein, it is believed the subject matter will be better understood from the following description of certain examples taken in conjunction with the accompanying drawings, in which like reference numerals identify the same elements and in which:
Cells were stimulated with 5 ug/ml M2339-IgG4 or IgG4 control for 48 hours. Only surface PD1 detection of anti-PD-1 VHH M-ICAP-T cells group is blocked by commercial PD-1 mAbs.
The promoter in each of the vectors shown is an EF1a promoter and a SV40 polyadenylation signal is used for transcription termination in both vectors. The expression constructs both include 5′ and 3′ ITR sequences.
Chimeric antigen receptor T cell (CAR-T) treatment technology is a field of immune cell therapy for cancer. CAR-T technology uses genetic engineering technology to splice, e.g. an antibody variable region gene sequence including at least one portion of the gene encoding a CDR portion of the antibody, with the intracellular region of a T lymphocyte immune receptor, and then to introduce the splice construct into a T cell by retrovirus or lentiviral vector, transposon or transfection. The expression cassette or a mRNA is transduced into lymphocytes and expresses the fusion protein on the cell surface, enabling T lymphocytes to recognize specific antigens in a non-MHC-restricted manner, enhancing their ability to recognize and kill tumors.
The structure of a Chimeric Antigen Receptor (CAR) was proposed by the Eshhar research team in Israel in 1989. Since then, it has been confirmed that T cells displaying a CAR-structured cell surface protein have a good effect in tumor immunotherapy.
The first generation of CAR receptors contained a single-chain variable fragment (scFv), and the intracellular activation signal was transmitted by a CD3ζ (CD3z) signal chain. However, first-generation CAR receptors lack a domain to provide a T cell costimulatory signal, which leads to the CAR-T cells only exerting transient effects, short survival time of the cells in the body and less secretion of cytokines. The second generation of CAR receptors introduces the intracellular domain of costimulatory signaling molecules, including, for example, CD28, CD134/OX40, CD137/4-1BB, lymphocyte-specific protein tyrosine kinase (LCK), inducible T-cell co-stimulator (ICOS), DNAX-activation protein 10 (DAP10) and other domains to enhance T cell proliferation and cytokine secretion. IL-2, IFN-γ and GM-CSF production increase, thereby breaking the immunosuppression of the tumor microenvironment, for example AICD (activation induced cell death (AICD)).
Third-generation CAR receptors recombine a secondary co-stimulatory molecule such as 4-1BB between the co-stimulatory structure CD28 and an ITAM signal chain, thus producing a triple-signal CAR receptor.
Engineered CAR-T cells have better effector function and survival time in vivo. Presently, the CAR structure commonly used in therapies is a second-generation CAR receptor, and its structure can be divided into the following four parts: an antibody single-chain variable region (scFv), a hinge region, a transmembrane region, and an intracellular stimulation signaling polypeptide. The CAR hinge region structure contributes to forming the correct conformation and forming a dimer. The length of the hinge region and the amino acid sequence characteristics contribute to determining the spatial conformation of the CAR and also affect the ability of the CAR to bind to tumor cell surface antigens.
Malignant lymphoma is divided into two categories: Hodgkin's lymphoma (HL) and non-Hodgkin's lymphoma (NHL). Hodgkin's lymphoma accounts for 10%-15% of lymphoma, while Non-Hodgkin's lymphoma is the fastest-growing malignancy in patients with onset. According to WHO statistics, there are currently about 350,000 new NHL patients in the world each year, and the death toll exceeds 200,000. B-cell lymphoma can be seen in both of Hodgkin's lymphoma and non-Hodgkin's lymphoma. Currently, clinical treatments for lymphoma include cytotoxic drugs such as glucocorticoids and alkylating agents, and targeted drugs based on specific molecular targets (such as rituximab, etc.), wherein combination chemotherapy based on targeted drugs significantly improves responses, clinical remission rate and cure rate of patients. However, there are still a large number of patients with lymphoma who are not sensitive to or have poor efficacy and are “real” refractory patients. Some new treatments (such as cellular immunotherapy) have relieved and prolonged survival in patients with partially relapsed or refractory lymphoma. There are many types of CAR-Ts currently being developed for hematological malignancies, including therapies using anti-CD19, anti-CD20, anti-Kappa light chain, anti-CD22, anti-CD23, anti-CD30, anti-CD70 and other antibodies to construct CAR-modified T cells. Antitumor studies have been conducted and in these anti-CD19 and anti-CD20 monoclonal antibodies were the most commonly used antibodies.
Choosing the right tumor antigen as a target is the key to designing a safe and effective CAR-T cell. Since CD19 is expressed only in normal and malignant B cells at various stages of differentiation and not on other non-B cells (such as hematopoietic stem cells), it is a potential target for the treatment of B-lineage tumors and a hot spot in CAR-T research. Thus, CD19CAR-T is widely used for malignancy such as acute B lymphocytic leukemia (B-ALL), chronic B lymphocytic leukemia (B-CLL), mantle cell lymphoma (MCL), NHL, and multiple myeloma (MM). A CD19CAR-T has been used in a clinical trial treatment of B cell lymphoma.
PD-1 (Programmed Death 1, reprogrammed cell death receptor 1) is a member of the regulatory T cell CD28 family and belongs to the immunoglobulin receptor superfamily. PD-1 and its ligand PD-L1/PD-L2 play important roles in the co-suppression and failure of T cells. Their interaction inhibits the proliferation of co-stimulatory T cells and the secretion of cytokines. The expression of the anti-apoptotic molecule BCL-xl impairs the function of tumor-specific T cells, leading to the inability of some tumor patients to completely eliminate the tumor. Anti-PD-1 antibody competes with the ligand PD-L1/PD-L2 for binding the PD-1 molecule of the tumor-specific T cell surface, thereby inhibiting complexation of PD-1 and PD-L1/PD-L2. This in turn overcomes the immune microenvironment inhibition caused by PD-1 complexation by PD-L1/PD-L2.
Currently commercialized anti-PD-1 antibodies are nivolumab and pidilizumab. These two monoclonal antibodies have been shown to have good clinical efficacy in solid tumors such as melanoma, colon cancer, prostate cancer, non-small cell lung cancer, and renal cell carcinoma. Recent clinical studies have confirmed that PD-1 antibody can be used in lymphoma therapy. However, anti-PD-1 antibodies still have some unavoidable problems in clinical applications. On the one hand, because anti-PD-1 monoclonal antibody is administered intravenously, most patients receiving PD-1 antibody blockade will have different degrees of drug administration side effects. Also, in vitro production of anti-PD-1 monoclonal antibody involves a complicated production preparation and purification process, which is costly and leads to expensive treatment.
In summary, CAR-T cells have the ability to kill tumor cells and can effectively enter the tumor tissue, but their activity is easily inhibited in the tumor microenvironment; and PD-1 antibody can reactivate the antitumor activity of T cells. However, conventional macromolecular antibodies or large fragments thereof have insufficient power to penetrate into solid tumors, and systemic drugs have large toxic side effects, and the cost of drugs is high.
Therefore, a solution to this problem is disclosed herein, in which an anti-PD-1 antibody can be efficiently expressed by maintaining the killing toxicity of CAR-bearing immune cells (e.g. a CAR-T cell), and the PD-1 antibody is expressed at a high level in or near the tumor by the CAR-bearing cell. This activity is expected to increase the tumor-killing efficacy of the CAR-bearing cells, while also reducing treatment costs.
Presently disclosed is a system having some features similar to CAR-T, but of a more generalized nature. Also, by including an extracellular (perhaps synthetic and of a not naturally-occurring amino acid sequence) peptide molecule having bispecific binding activity of binding both an effector cell bearing a CAR and a target cell bearing a cell surface antigen as an “Immune Cell Activator Polypeptide” (ICAP), the activity level of the CAR-bearing effector cells can be modulated by control of the amount of the ICAP available to bind the CAR. Such a system can be applied to solve the problem of high tonic activity exhibited by CAR-T cells of the prior art.
Some of the terms related to the present disclosure are explained below.
In the present disclosure, the term “expression cassette” refers to the entire element required for expression of a gene, including a promoter, a coding sequence, and a polyA tailing signal sequence.
The term “coding sequence” is defined herein as a portion of a nucleic acid sequence that encodes the amino acid sequence of a polypeptide product (eg, a CAR, a Single Chain Antibody or a domain thereof). The boundaries of the coding sequence are typically determined by a ribosome binding site (for prokaryotic cells) immediately upstream of the open reading frame of the 5′ end of the encoded mRNA and a transcription termination sequence immediately downstream of the open reading frame of the 3′ end of the encoded mRNA. A coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.
The term “Fc”, (fragment crystallizable) is a portion of a mammalian antibody, and refers to a peptide located at the end of the handle of the “Y” structure of the antibody molecule, comprising the CH2 and CH3 domains of the heavy chain constant region of the antibody, and is the site of many molecular and cellular interactions that provide some of the biological effects of a mammalian antibody.
The term “costimulatory molecule” refers to a molecule that is present on the surface of an antigen presenting cell and that binds to a costimulatory molecule receptor on a Th cell to produce a costimulatory signal. The proliferation of lymphocytes requires not only the binding of antigens, but also the signals of costimulatory molecules. The costimulatory signal is transmitted to the T cells mainly by binding to the co-stimulatory molecule CD80 on the surface of the antigen presenting cells, and CD86 binds to a CD28 molecule on the surface of the T cell. B cells receive a costimulatory signal that can pass through a common pathogen component such as LPS, or through a complement component, or through activated antigen-specific Th cell surface protein CD40L.
The term “linker” is a polypeptide fragment that links between different proteins or polypeptides for the purpose of maintaining the spatial relationship of the linked proteins or polypeptides to maintain the function or activity of the protein or polypeptide, for example by relieving steric inhibition of binding of a ligand. Exemplary linkers include linkers containing glycine and/or serine, as well as, for example, a Furin 2A peptide.
The term “specifically binds” refers to the reaction a binding protein and a ligand, for example as between an antibody or antigen-binding fragment and the antigen to which it is directed. In certain embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific for an antigen) means that the antibody-antigen affinity is characterized by a binding constant, Kd, of less than about 10-5 M, such as less than about 10−6 M, 10−7 M, 10−8M, 10−9 M or 10−10 M or less. “Specifically recognizes,” or “specific recognition” has a similar meaning.
The term “pharmaceutically acceptable excipient” refers to carriers and/or excipients that are compatible pharmacologically and/or physiologically to the subject and active ingredient, which are well known in the art (see, for example, Remington's Pharmaceutical Sciences, editor Gennaro A R, 19th ed. Pennsylvania: Mack Publishing Company, 1995, hereby incorporated by reference in its entirety and for all purposes), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers. For example, pH adjusting agents include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride.
The term “effective amount” refers to a dose that can achieve a treatment, prevention, alleviation, and/or alleviation of a disease or condition described herein in a subject.
The term “disease and/or condition” refers to a physical state of the subject that is associated with the disease and/or condition described herein.
The term “subject” or “patient” may refer to a patient or other animal that receives the pharmaceutical composition of the invention to treat, prevent, ameliorate and/or alleviate the disease or condition of the invention, particularly a mammal, such as a human, a dog, monkeys, cattle, horses, etc.
As used herein, a “chimeric antigen receptor” (CAR) is an artificially engineered protein that binds a specific molecule, for example a tumor cell surface antigen, and that stimulates a proliferative program in an immune cell-type effector cell. A CAR typically comprises, in order from amino- to carboxy-terminus, an optional signal peptide (which might be removed during the process of localization of the CAR in the cell membrane of the host cell); a polypeptide that specifically binds to another protein (“label domain”), such as an antigen binding region of a single chain antibody; an optional (but typically present) hinge region; a transmembrane region; and an intracellular signaling region (see, e.g.
In the present application, a “VHH domain” can refer to a variable domain of a single heavy-chain antibody (“VHH antibody”), such as a camelid antibody. A “Single Chain Antibody” (SCA) is a single chain polypeptide and typically includes a number of relatively conserved domains that associate together as the polypeptide folds to form a Framework Region (FR region), and variable regions that associate together to form a variable, antigen-binding domain. A VHH antibody is accordingly a kind of a SCA. In accordance with this terminology, a variable domain present in a naturally occurring single heavy-chain antibody, will also be referred to herein as a “VHH domain”, in order to distinguish such from the heavy chain variable domains that are present in conventional 4-chain antibodies (which will be referred to herein as “VH domains”) and from the light chain variable domains that are present in conventional 4-chain antibodies (which will be referred to herein as “VL domains”).
Isolated single variable domain polypeptides preferably are ones having the full antigen-binding capacity of their cognate SCAs and are stable in an aqueous solution.
Stable, antigen-binding single chain polypeptides comprising one or more domains (of either FR or variable region origin) derived from, or similar to, domains of mammalian antibodies, such as a VH domain, are also encompassed by “Single Chain Antibodies” herein.
A “nanobody” can include a SCA or VHH antibody, or one or more domains thereof, but this word is used more typically to describe an engineered polypeptide comprising one or more VHH domains, and optionally further comprising one or more FR domains, and can additionally or alternatively further comprise additional stable domains that have some further biological activity, such as binding to a flurorophore or binding and activating an extracellular receptor.
Disclosed herein is a novel cell therapy product, an engineered immune effector cell, which can be one such as a so-called “Baize Super Cell,” comprising a chimeric receptor, that can be induced to express a secreted protein in a controllable manner and in situ.
In some embodiments, the engineered immune effector cell constitutively expresses high levels of an effector polypeptide, such as a single chain anti-PD-1 antibody (VHH-PD-1). In some such embodiments, T-cell proliferation activated by binding of a “label domain” of a of an immune effector cell that is a T-cell by a cell surface-associated antigen of a cell provides a very large number of T cells that constitutively secrete an effector polypeptide. In instances where the label domain is bound by an antigen on the surface of a tumor cell, an immune modulatory effector polypeptide can be one that is constitutively expressed and, by virtue of being secreted in the vicinity of the tumor cell, alleviates or avoids immunotolerance induced by e.g. PD-1:PD-L1/L2 complex formation.
Additionally or alternatively, an engineered effector cell as disclosed herein can be engineered to comprise a nucleic acid vector that comprises a coding sequence construct encoding one or more “effector polypeptides” that is expressed under control of a promoter that is operable in an immune cell and that further comprises transcription termination sequences operable in an immune cell. The promoter can be a constitutive promoter, such as a EF1a promoter or a CMV promoter.
The nucleic acid vector can be a retroviral vector or a lentiviral vector. The nucleic acid vector can be a DNA or RNA vector. The vector can comprise a PiggyBac (PB) transposon or a SleepingBeauty (SB) transposon or portion thereof. The vector can comprise transposon-specific Inverted Terminal Repeat sequences, which are typically located at both ends of a transposon-based vector.
An engineered effector cell as disclosed herein can be one in which either or both of an expression cassette encoding an ICAP and an expression cassette encoding one or more effector polypeptides are integrated into the nuclear genome of the effector cell.
A protein to be secreted by the effector cell can be one that is immunostimulatory, such as a polypeptide that specifically binds 4-1BB or OX40, or immuno-inhibitory (for example so as to treat an allergy response or an arthritic condition), such as a polypeptide that specifically binds TNF-α or IL-6.
A preferred protein to be secreted by the effector cell is an antibody or a fragment thereof, or a polypeptide that is a single chain-single domain polypeptide, for example a VHH nanobody or scFv protein. One class of proteins that can be secreted is an immune checkpoint receptor antagonist or agonist antibody with or without a Fc domain. However, other proteins might be expressed and secreted by an engineered effector cell, such as cytokines or another immunomodulatory protein. For example, an antibody, an antigen-binding portion of an antibody or a single chain antibody, such as a VHH nanobody, against PDL1, CTLA-4, CD-40, LAG-3, TIM-3, BTLA, CD160, 2B4, CD40, 4-1BB, GITR, OX-40, CD27, HVEM or LIGHT can be expressed and secreted from an effector cell. Examples of secreted cytokines from an effector cell may include TGF-β, VEGF, TNF-α, CCR5, CCR7, IL-2, IL-7, IL-15 and IL-17. An engineered effector cell of the present invention can express and secrete two or more different types of effector polypeptides, including different antibodies, cytokines, or combinations thereof. As an example, an engineered effector cell can secrete an anti-PDL1 antibody and an anti-CTLA-4 antibody, or an anti-PDL1 antibody and VEGF antibody.
An example of a secreted effector protein is an anti-PD-1 VHH antibody (1182) having an amino acid sequence
The host immune cells that are engineered can be various T-cells, CIK (cytokine induced killer cells), DC-CIK (dendritic cell/CIK), NK (natural killer cells), NKT (natural killer T cells), stem cell, TIL (tumor infiltrating lymphocytes), macrophage and other immune cells. The host immune cells are typically autologous cell of a subject being treated for a disease.
In some embodiments, the engineered immune cells are transformed with a vector comprising a coding sequence construct having at least 3 structural components: a polynucleotide encoding a first domain that includes intracellular signaling domains that activate a transcriptional program in an “activated” T cell, for example, a CD3ε (CD3e) or a CD3ζ (CD3z) domain of a T-cell surface glycoprotein; a second polynucleotide encodes a domain that contains a transmembrane domain (and optionally spacer peptides), for example one from a CD28 protein; and third polypeptide that encodes a domain that is a “label” polypeptide, specific binding of which by another polypeptide activates a transcriptional program in a host immune cell, such as a T cell via the intracellular signaling domain.
The intracellular signaling domain can include domains that participate in immune co-stimulatory signaling (for example a B7 binding domain), and additionally or alternatively an ITAM domain of a CD3e. Preferably an ITAM domain includes an amino acid sequence YMNM (SEQ ID NO:4).
In some embodiments, both of a transmembrane domain and an intracellular signaling domain are those of a CD28 protein.
In some embodiments, the signal transduction domain comprises an immune co-stimulatory domain joined to a CD3e domain, such as CD28/CD3e, 4-1BB/CD3e, ICOS/CD3e, CD27/CD3e, OX40/CD3e or CD40L/CD3e.
The label domain polypeptide is preferably one that is not expressed, or is minimally expressed, in adult human tissues. For example, the label polypeptide might be derived from a protein that only expressed, or expressed predominantly, in embryonic human cells (i.e., a “fetoprotein”) or a label polypeptide can be a completely synthetic amino acid sequence.
Examples of fetoproteins from which a label polypeptide might be derived include fetoproteins expressed during embryogenesis such as Oct-4, Sox-2, and Klf-2. In some embodiments, less than the complete full-length protein is used; typically between 20-100 aa long polypeptides are used. Below are the amino acid sequences for Oct-4, Sox-2 and Klf-2:
A label domain portion of an ICAP can be a polypeptide having an amino acid sequence MAGHLASDFAFSPPPGGGGDGPGGPEPGWVDPRTWLSF (SEQ ID NO:8).
The ICAP label domain may contain a structurally inert domain from human mesothelin ECD. For polypeptides encoding the mesothelin domain, it may contain peptide sequences from domain I, II or III as follows:
A label polypeptide can be derived from a structural membrane protein that has no intracellular signal transduction function or interaction with other bioactive molecules, to provide a “structurally inert” domain of the structural membrane protein provided it is one that is normally bound by another protein or carbohydrate and so the epitope constituting the label domain is not exposed to antibodies in vivo. The label polypeptide preferably has little or no immunogenicity. Immunogenicity of the label polypeptide can be determined by 1) in silico computational algorithms on the number of T-cell epitopes 2) in vitro assays to determine T cell activation potential, and 3) in vivo experiments using animal models.
Any label domain as described above can be combined with any transmembrane domain described above and any intracellular signaling domain as described above to form an ICAP polypeptide. Short polypeptide linkers can be used to join domains of an ICAP.
For example, any of the label domains described above can be encoded as the “label domain” portion of the plasmid pNB338B-ICAPs-VHH shown in
The construct is expressed in the effector cell to produce a “immune cell activating polypeptide” (ICAP), that localizes to the outer cell membrane such that the label domain is extracellular.
An effector cell as disclosed herein can be used with a bispecific polypeptide—that is, polypeptide having two functional domains joined by a joining polypeptide, or by chemical conjugation, each domain having activity of specifically binding a different ligand. Herein, in some embodiments, the bispecific polypeptide is also called a “VHH-TCP”, as a preferred form for the bispecific polypeptide comprising two or more single chain nanobodies (single chain, single domain antibodies).
One domain of the bispecific polypeptide comprises an amino acid sequence that specifically binds to the label domain of an ICAP on the surface of an effector cell (L-bd), and one domain of the bispecific polypeptide specifically binds to a protein on the surface of a “target”, which is preferably a cellular target, such as a tumor cell, but might be any cell or surface bound with the target protein (CSP-bd). Such a surface presented target polypeptide is called herein a “cell surface protein” or an epitope thereof.
Such cell surface proteins can be antigens associated with tumors, auto-immunity disease, or cellular or organismic senescence; e.g. CD19, mesothelin, BCMA, EGFR, vimentin, Dcr2 or DPP4. In some embodiments, the target cells are cells that abnormally express one or more of these proteins, either as to amount or as to a mutated protein; e.g. a tumor of a B cell, mesothelial cell, breast cell, or fibroblast cell.
The bispecific polypeptide (VHH-TCP), as used herein, can include additional domains, to provide additional binding, or biochemical or physiological activities, for example to recognize multiple epitopes either from the same target protein, or epitopes from multiple target proteins (a “polyspecific polypeptide”, which includes, for example, a tri specific, tetraspecific, pentaspecific or hexaspecific polypeptide). The bispecific polypeptide can also include one or more binding motifs to recognize a human IgG Fc domain as a label domain of an ICAP to effect an effector cell activity-switcher through ADCC, CDC and ADCP mechanisms.
Additionally or alternatively, the bispecific (polyspecific) polypeptide (VHH-TCP) can also include one or more domains derived from serum albumin with various molecular weights to control the half-life of the bispecific polypeptide in vivo.
A domain for binding a fluorophore can be included in a bispecific polypeptide to allow tracking in vivo (e.g. by examination of fluorophore-stained tissue samples) of the bispecific polypeptide and of cells to which the bispecific polypeptide is specifically bound.
Preferably, the domains of the bispecific polypeptide can be joined to one another N-terminal to C-terminal by one or more linker peptides. The length of linkers can be adjusted to tune the molecular weight of the bispecific polypeptide or steric interactions among its domains (e.g. to lessen them).
Linker portions of a bispecific polypeptide can also include amino acid sequences that are susceptible to cleavage by peptidases in the blood, thereby limiting the half-life of the bispecific polypeptide in the blood or extracellular matrix. For example, the amino acid sequences RVLAEA (SEQ ID NO:12), EDVVCCSMSY (SEQ ID NO:13) and GGIEGRGS (SEQ ID NO:14) are cleavable by matrix metalloproteinase-1, and the amino acid sequence VSQTSKLTRAETVFPDV (SEQ ID NO:15) is cleavable by Factor IXa/Factor VIIa.
In some embodiments, one or more, e.g. all, of the active domains are comprised of VHH nanobody polypeptides.
A L-bd can be a single antibody domain derived from the VHH domain of a camelid IgG. The CDR3 region of such a VHH domain can contain 15-20 amino acids that serve as the paratope binding to epitope(s) on the label domain.
The bispecific polypeptide can comprise a L-bd that is a VHH domain that specifically binds a label polypeptide and a CSP-bd that is a VHH domain that specifically binds to CD19 or CD20. Such a bispecific polypeptide would be useful in treating a B-cell lymphoma, such as a non-Hodgkin's lymphoma. In some embodiments, a bispecific polypeptide can comprise a L-bd that is a VHH domain that specifically binds a label polypeptide and a CSP-bd that is a VHH domain that specifically binds to EGFR. An amino acid sequence from the CDR3 region of the VHH antibody can bind to EGFR of on the surface of non-small cell lung cancer cells. Such bispecific polypeptides would be useful in treating a non-small cell lung cancer.
A bispecific polypeptide can comprise a L-bd that is a VHH domain that specifically binds a label polypeptide and a CSP-bd that is a VHH domain that specifically binds to CPC3. In some embodiments, a bispecific polypeptide can comprise a L-bd that is a VHH domain that specifically binds a label polypeptide and a CSP-bd that is a VHH domain that specifically binds to BCMA. In some embodiments, a bispecific polypeptide can comprise a L-bd that is a VHH domain that specifically binds a label polypeptide and a CSP-bd that is a VHH domain that specifically binds to HER2. Such a bispecific polypeptide would be useful in treating a HER2+ breast cancer tumor.
An exemplary bispecific polypeptide that comprises two VHH domains joined by a linker (VHH that binds to a label domain containing a structurally inert peptide derived from the human mesothelin's ECD+linker+anti-EGFR VHH) has an amino acid sequence
Preferably binding of the bispecific polypeptide to epitopes on other cells than on the target cells does not have significant impact on the pharmacokinetics or pharmaco-distribution of the bispecific polypeptide in vivo, and preferably such binding as does occur does not cause any significant observable physiological effects other than activating the effector cell expressing the associated label domain to be bound by the bispecific polypeptide.
By virtue of the embodiments illustrated and described herein, Applicant has devised a method and variations thereof for treating tumors using the engineered effector cells and bispecific polypeptides disclosed herein.
In one such method the engineered immune cells, which can be T-cells, or other cells types as described herein as effector cells, are injected directly into a solid tumor. Alternatively, the engineered immune cells can be administered intravenously (IV, e.g. when a leukemia or lymphoma is treated). Different administration methods may be performed depending on the disease indication. In most cases, IV administration is performed to treat disease. Intraperitoneal administration can be performed to treat malignant pleural mesothelioma (MPM).
For treatment of a solid tumor, direct injection into the tumor is expected to result in better distribution of cells within the tumor microenvironment (higher amounts of the engineered immune cells in proximity to the target tumor cells).
In a typical treatment method, the amount of a VHH-TCP to be administered can range from 10 ng/ml to 100 ng/ml together with the engineered immune cells at a concentration of, e.g. 5×104, 1×105, 5×105, or 1×106 engineered cells/ml.
In one example embodiment of a treatment method, which does not utilize a VHH-TCP activating molecule, the engineered immune cells are T cells expressing an ICAP having a VHH label domain that specifically binds to CD19 on a B cell and having the transmembrane domain and intracellular signaling domains of the common T-Cell Receptor (i.e. CD28 and CD3e). The engineered T-cells also comprise a vector for expression of an anti-PD-1-Fc effector polypeptide under control of a constitutive promoter. After administration of the cells to a subject, the label-VHH domain of the ICAP specifically binds to CD19 on B cells, and the binding transduces signals to the engineered immune cells which are then activated upon CD3 and CD28 intracellular signaling and proliferate in the vicinity of the B cell target. The proliferating cells secrete a large amount of the anti-PD1 effector protein in the vicinity of the bound B cell.
The disclosed system, in its various embodiments, provides one or more of the following advantages. Not every embodiment will exhibit every one of the advantages set out below.
In embodiments of the ICAP label domain, a polypeptide derived from fetoprotein or structural membrane proteins provides a wide range of possible L-bds for a VHH-TCP binding, and may improve cell therapy safety due to no or less immunogenicity of the domain.
The diversity of domains that can be included in bispecific polypeptide (VHH-TCP) provides the ability to alter many properties such as VHH-TCP affinity to the effector cell, and the range of cells that can be targeted by the CSP-bd is broad. Also other functional domains can be added, and epitope binding valency can be adjusted to efficacy and safety of use of the system to treat a disease.
A bispecific polypeptide (VHH-TCP) that specifically binds to a label domain of an immune cell activator polypeptide that includes a signaling domain that activates proliferation of the immune cell host and specifically binds to a cell surface protein of B cells such as the CD19 ligand, can induce proliferation of the effector cells in vivo increase the population of the immune effector cells, thus increasing the amount of the effector polypeptide, in the B cell binding vicinity. This allows for time savings and avoidance of the costs of in vitro production of the effector polypeptide.
A bispecific polypeptide (VHH-TCP) can be engineered in various formats to optimize the effector cell activity through the length and flexibility of linkers among VHHs in the bispecific polypeptide (VHH-TCP), the position of each binding motif and the overall size of VHH-TCM.
Effector cell activity in vivo can be controlled by dosing with different amounts of a bispecific polypeptide (VHH-TCP), and/or controlling the half-life of a VHH-TCP. This is a novel and comprehensive approach to minimize the toxicity of a CAR-based therapy.
Furthermore, using an appropriate label protein that specifically binds Fc epitopes, effector cells can be activated by ADCC effects.
Importantly, the characteristics of a nanobody such as small size, high stability and easy engineering offers unique advantages for optimizing a treatment system in vivo.
Discontinuation of VHH-TCP administration to a subject can prevent adverse effects associated with the persistent effector cell activity while also providing the opportunity for subsequent VHH-TCP administration in the event of disease relapse.
In situ secretion of antibody, preferably nanobody, by activated effector cells can either inhibit or stimulate immune checkpoint receptors to improve targeting of solid tumors through TME (tumor microenvironment) penetrance, proliferation and persistence. Nanobody bispecific polypeptides have advantages over conventional antibodies in penetrating the TME due to their small size and great stability.
The effector cell—bispecific polypeptide (VHH-TCP) system disclosed herein can improve upon many of the pitfalls that accompany current CAR-T therapy: for instance by targeting multiple tumor antigens with a single, standardized immune receptor, and diverse VHH-TCP structures can be used to control and optimize immune cell activity. Treatments utilizing the disclosed system are expected to exhibit less toxicity or side-effects. Further, the components of the system are easily and cost effectively manufactured. The diversity of ligands and binding domains that can be incorporated into the ICAP and bispecific polypeptide (VHH-TCP) allows a modular system to be used to treat a broad variety of diseases or to conduct research, for example incorporating a FITC-binding domain into a VHH-TCP allows the fate of activated effector cells to be followed in vivo.
1. Generation of Modified Effector T Cells by Electroporation
An ICAP comprises of a label polypeptide (27 aa of mesothelin, or fetoproteins), a CD28 transmembrane domain, a CD28 intracellular co-stimulatory signaling domain (CD28IC) and a CD3ζ. The 1182-Fc(EQ) comprises of VHH-1182 and IgG4 Fc domain.
A 1182-Fc(EQ) structural gene is cloned into the piggyBac transposon vector pS338B to obtain a plasmid pS338B-1182-Fc(EQ) (
Human Peripheral blood mononuclear cells (PBMCs) of healthy donors are purchased from AllCells (Shanghai, China). PBMCs are cultured in AIM-V medium supplemented with 2% fetal bovine serum (FBS; Gibco, USA) at 37° C. in a 5% CO2 humidified incubator for 0.5-1 hr, and then harvested and washed twice using Dulbecco's phosphate-buffered saline (PBS).
PBMCs are counted and electroporated with 6 μg pNB338B-ICAP-VHH plasmid or an equal quantity of MOCK plasmid in electroporators (Lonza, Switzerland) using a Amaxa® Human T Cell Nucleofector® Kit according to the manufacturer's instructions. Thereafter, T cells transfected with ICAP-VHH/1182-Fc (EQ) plasmids or MOCK/1182-Fc(EQ) plasmids are specifically stimulated in 6-well plates, which are coated with anti-CD3 antibody/anti-CD28 antibody (5 μg/mL), for 4-5 days. Transformed T cells are then cultured in AIM-V medium containing 2% FBS and 100 U/mL recombinant human interleukin-2 (IL-2) for 10 days to generate a sufficient quantity of effector T cells.
2. Transduction Efficiency Assay
Transduction efficiency of label polypeptide into T cells is determined by flow cytometry using biotin-conjugated anti-IgG4(Fc) antibody and a PE-conjugated streptavidin secondary antibody.
3. Binding Efficiency Assay
The binding of bispecific polypeptide to common T cells is measured by flow cytometry using anti-CD19-PE antibodies. The ratio of cells positive for CD19 and label (e.g. meso) is compared to determine the binding efficiency.
4. Proliferation Ability Assay (in Culture with Bispecific VHH and Tumor Cells)
1×107 transformed T cells are prestained with carboxyfluorescein succininmidyl ester for 10 minutes and recovered in medium for another 10 minutes. 5×105 cells are counted and cocultured with tumor cell lines that express different antigens, including BCMA, EGFR, Mesothelin, MUC1 and GPC3, as well as bispecific VHH for 7 days, replacing the culture medium every 3-4 days with fresh medium (AIM-V+2% FBS). The effector cells are then assayed for proliferation by flow cytometry.
5. Quantification of 1182-Fc-VHH Secretion
5×105 cells are seeded at in 6-well plates with 1 ml medium, and tumor cells and bispecific VHH are added and cocultured for 48 hours. Then the effector T cell suspension is centrifuged at 3000 rpm for 3 min; the supernatant is retained and 1182-Fc protein is quantitatively detected by ELISA.
6. Cytotoxicity Assay (for Adhesion Cell Lines)
The cytotoxicity of effector T cells transduced with label construct or vector control is determined by using an impedance-based xCELLigence RTCA TP Instrument.
Target tumor cells are seeded in a resistor-bottomed 96-well plate at 10,000 cells per well within the RTCA TP instrument overnight (more than 16 hours). The bispecific VHH antibody is added to the cultured target tumor cells and the cells are further cultured for 30 minutes. Then transformed T cells harboring the plasmids pS338B-1182-Fc(EQ) and pNB338B-ICAP-VHH (effector cells) are incubated with target tumor cells at different effector cell:target cell ratios for about 100 hours (the end point depends on the killing efficiency of transformed T cells). During the experiment, the cell index values are closely correlated with tumor cell adherence, such that lower cell attachment indicates higher cytotoxicity, and are collected every 5 minutes by the RTCA system and an EnVision® Multilabel Plate Reader (PerkinElmer). The real-time killing curves are automatically generated by the system software. Specific lysis (%) of each transformed T cell are also calculated using the data of the end point [specific lysis=(cell index of tumor cells alone−cell index of transformed T cells cocultured with tumor cells)/cell index of tumor cells alone].
7. Cytotoxicity Assay (for Suspension Cell Lines)
The cytotoxicity of effector T cells transduced with label construct or vector control is determined according to the manufacturer's protocol (DELFIA® EuTDA Cytotoxicity Reagents AD0116—PerkinElmer). Briefly, target tumor cells are washed with PBS and fluorescence enhancing ligand and are incubated for 5-30 minutes at 37° C. 100 ul of target cells (10,000 cells) are placed into a V-bottom plate containing bispecific polypeptide that specifically binds to both of the target tumor cells and to the effector cells (that is, the transformed T cells), and 100 ul of effector cells are added with varying cell concentration. 20 ul of the supernatant were transferred into 200 μL of Europium Solution following 15 minutes incubation at room temperature. The fluorescence is measured in the time-resolved fluorometer. Specific release (%)=Experimental release (counts)−Spontaneous release (counts)/Maximum release (counts)−Spontaneous release (counts)×100.
8. Specific Targeting Activity In Vivo
NOD-SCID IL2 Ry−/− (NSG) mice are raised under the pathogen-free condition (Shanghai, China). The animal experiments are approved by Institutional Animal Care and Use Committee (IACUC). To establish xenograft tumor models, NSG mice are subcutaneously inoculated with 5×106 EGFR+ lung tumor cells and 5×106 MSLN+ ovarian tumor cells mixed with an equal volume of Matrigel™. The tumor dimensions are obtained using vernier calipers, and the tumor volumes are calculated based on the formula: V=½(length×width2). When the tumor burdens are approximately 100 mm3, the Fluc-labelled effector T cells and bispecific EGFR-targeting VHH are intravenously injected. The specific targeting of effector T cells to lung is confirmed by bioluminescence imaging (BLI). On day 5, another bispecific MSLN-targeting VHH is intravenously injected to observe the specific targeting of effector T cells to ovarian tumor cells. The proliferation ability of effector T cells in vivo is monitored by bioluminescence imaging using a Xenogen IVIS imaging system (PerkinElmer, USA).
9. Proliferation and Antitumor Activity In Vivo
To establish xenograft tumor models, NSG mice are subcutaneously inoculated with 5×106Fluc-labelled tumor cells mixed with an equal volume of Matrigel™. When the tumor burdens are approximately 100 mm3, mice are randomly separated into three groups (5 mice per group) for intravenous injection (i.v.) of MOCK-T, effector T cells, or PBS vehicle with polypeptides VHH, the time point of which is designated as day 0.
Peripheral blood of all mice is taken from the tail vein to detect the proliferation of effector T cells and copy numbers of ICAP gene. Mice are euthanized after a moribund state is reached, and then the bone marrow, blood and spleen are collected. The percent of CD3+ T cells in the above tissues and the memory T cell subsets in spleens are analyzed by flow cytometry. During the whole experimental progress in vivo, mice body weight is measured using an electronic balance. The tumor progression is confirmed by bioluminescence imaging (BLI) using a Xenogen IVIS imaging system (PerkinElmer, USA). All measures are conducted every five days.
10. Hematoxylin-Eosin (H&E) Staining and Immunohistochemistry (IHC)
H&E and immunohistochemistry are performed to evaluate the safety of the cell therapy. Mouse tissues (heart, liver, spleen, lung, kidney and brain) are fixed with formalin and then embedded with paraffin. The tissues are cut into 4 μm thickness consecutively using a R1\42245 microtome (Leica, Germany), and then stained with H&E. To detect the infiltrative ability of the effector T cells within tumor tissues, IHC analysis is performed using anti-CD3 antibody (Abeam, #ab16669) at 1:100 dilution. Images are taken with an AXIOSTAR PLUS microscope (ZEISS, Germany).
11. Tissue Distribution Assay
The tissue distribution of 1182-VHH, transfected T cells and adaptor VHH proteins is determined using Quantitative Real-time PCR (RT-qPCR). Mouse tissues (heart, liver, spleen, lung, kidney and brain) are digested to prepare single cell suspensions. Total DNA is extracted from T cells using Genomic DNA Extraction Kit Ver. 5.0 (TAKARA, China) according to the manufacturer's instruction. Real-time polymerase chain reaction is performed using TaqMan™ Universal Master Mix II (ThermoFisher Scientific, USA). Primers and probes of CAR and actin are synthesized or labeled by Shanghai Generay Biotech Co. Ltd (Shanghai, China). Quantitative real-time PCR reaction is performed in two steps: (1) Pre-incubation: 95° C. for 5 minutes; (2) Amplification: 40 cycles of 95° C. for 20 seconds followed by 60° C. for 1 minute. All reactions are performed in triplicate.
12. Statistical Analysis
All data are presented as mean±SD. T-test is used to evaluate differences between two independent groups. One-way ANOVA is used to compare whether there are any statistically significant differences between three or more independent groups. Two-way ANOVA is used to determine the effect of two nominal predictor variables on a continuous outcome variable. All statistical analysis is performed using Graphpad Prism 7 version software (La Jolla, Calif.). All data with error bars are presented as mean±SD. Statistics significant difference is considered as follows: P≥0.05 (ns), P<0.05 (*), P<0.01 (**), P<0.001 (***), P<0.0001 (****).
The identification and characterization of one specific VHH nanobody towards MSLN, BCMA or EGFR with high affinity using an alpaca immune library is described.
1. VHH Nanobody Towards MSLN
For the first immunization, 400 μg MSLN-hFc emulsified using Freund's complete adjuvant was subcutaneously administered to each alpaca. Two weeks later, 200 μg MSLN-hFc emulsified using Freund's incomplete adjuvant was subcutaneously administered. After that, 5 additional immunizations were carried out with 200 μg MSLN-hFc emulsified using Freund's incomplete adjuvant every other week. High serum titer against both MSLN-His antigen as well as HEK293T-MSLN stable cell line was confirmed by ELISA and FACS.
Seven days after the last injection 50 mL of blood were collected, lymphocytes were purified from the sample, RNA was extracted and used for immune library construction. Two rounds of solid protein panning were conducted with MSLN-His antigen followed by ELISA screening and FACS verification. One positive clone named M2339(VHH) was obtained.
The antibody was expressed as a hFc fusion protein, named as M2339(VHH) with the procedure that was described in the patent publication WO2020176815A2 (hereby incorporated by reference in its entirety and for all purposes). The binding affinity of M2339(VHH) towards MSLN antigen was tested by surface plasmon resonance (SPR). First, M2339(VHH) was passed through the sensor chip with protein A immobilized in advance, and the antibody was captured by protein A. Then, five different concentrations of MSLN-His protein were used as the mobile phase, and the binding time and dissociation time were 30 min and 60 min, respectively. Biacore evaluation software 2.0 (GE) was used to analyze the on-rate (kon), off-rate (koff) and equilibrium constant (KD). As shown in Table 1 below, the affinity of M2339(VHH) towards MSLN antigen was high with the KD of 2.64E-10.
The binding affinity of M2339(VHH) towards HEK293T-MSLN cells was identified with flow cytometry. One 96-well plate was incubated with HEK293T cells and HEK293T-MSLN cells in different wells at 3×105 cells per well, then serially diluted M2339(VHH) was incubated for half an hour, after that the detection secondary antibody anti-human IgG PE (Jackson Immuno Research, Code: 109-117-008) was incubated before detection with CytoFLEX flow cytometer. “Isotype” is an isotype control (negative control). As shown in
2. VHH Nanobody Towards BCMA
This example describes the identification and characterization of one specific VHH nanobody towards BCMA with high affinity using an alpaca immune library. The procedures for alpaca immunization, blood collection, library construction, solid panning, ELISA or FACS screening for positive clones, antibody purification and followed antibody characterization by SPR and FACS are described above in Example 2. One positive clone named as B029(VHH) was obtained.
As shown in Table 1, the affinity of B029(VHH) towards BCMA-His antigen was high with a KD of 1.25E-10.
As shown in
3. VHH Nanobody Towards EGFR
This example describes the identification and characterization of one specific VHH nanobody towards EGFR with high affinity using an alpaca immune library. The procedures for alpaca immunization, blood collection, library construction, solid panning, ELISA or FACS screening for positive clones, antibody purification and antibody characterization by SPR and FACS are described above in Example 2. One positive clone named as E454(VHH) was obtained.
As shown in Table 1, the affinity of E454(VHH) towards EGFR His antigen was high with the KD of 1.27E-09.
As shown in
This example described the identification and characterization of the label used in this invention that was recognized M2339(VHH) with a similar affinity binding to mesothelin.
Different mesothelin ECD domain with human Fc were expressed in 293T cells and purified by protein A column. Affinity was determined by SPR. Protein A chip was used to capture different antigens, various concentrations of M2339(VHH) were injected at a flow rate of 10 μl/min with an association time of 120 to 180 seconds and a dissociation time from 180 to 1200 seconds. Binding kinetics were determined using Biacore Evaluation software in a 1:1 fit model.
As shown in
These results demonstrated that M2339 bound mesothelinII+III with a high affinity and could be used as an adaptor VHH, mesothelinII+III can be used in an ICAP in T cells.
To generate so-called M-ICAP-T, for activating immune cells, e.g. T cells, different vectors were constructed as shown in
All vectors were transfected into 293T cells with Lipofectamin2000 (ThermoFisher, USA) and after 2-4 days flow cytometry was used to test their expression rate. For flow cytometry detection, a 19R73-CD19CAR and GFP vector was used to prepare a blank cell control, M2339-hFc and biotin conjugated anti-His mAb were used as primary antibody, fluorophore-conjugated anti-human Fc and fluorophore-conjugated streptavidin were used as secondary antibody. As shown in Table 3 and
M-ICAP expression vectors containing different signal peptides (SP-MSLN, SP3, SP5) were constructed and fused with the T cell activation/signal transduction domain (CD28/4-1BB, CD3ζ) of traditional CAR vector, as shown in
Human Peripheral blood mononuclear cells (PBMCs) of healthy donors were purchased from AllCells (Shanghai, China). PBMCs were cultured in AIM-V medium supplemented with 2% fetal bovine serum (FBS; Gibco, USA) at 37° C. in a 5% CO2 humidified incubator for 0.5-1 hr, and then harvested and washed twice using Dulbecco's phosphate-buffered saline (PBS). PBMCs were counted and electroporated with 6 μg M-ICAP, SP3-M-ICAP and SP5-M-ICAP vectors in electroporators (Lonza, Switzerland) using an Amaxa® Human T Cell Nucleofector® Kit according to the manufacturer's instructions. Thereafter, transfected T cells were stimulated specifically in 6-well plates coated with anti-His/M2339 (VHH-Fc) plus anti-CD28 antibody (5 μg/mL), for 4-5 days, then cultured in AIM-V medium containing 2% FBS and 100 U/mL recombinant human interleukin-2 (IL-2) for 10 days to generate a sufficient quantity of effector T cells. Transduction efficiency of label polypeptide (M-ICAP expression) on T cells was determined by flow cytometry using biotin-conjugated anti-His antibody and a PE-conjugated streptavidin secondary antibody.
As shown in
M-ICAP were fused into several different CAR sequences, and obtained M-ICAP-T cells were obtained by electroporation combined with specific activation using donor derived PBMC cells. The ICAP vector comprised a label polypeptide (from mesothelin II+III domain), a CD28 transmembrane domain, a CD28/4-1BB intracellular co-stimulatory signaling domain (CD28/4-1BBIC) and a CD3ζ domain. The 1182-Fc(EQ) comprises VHH-1182 and IgG4 Fc domains.
Generation of cells expressing an ICAP CAR (ICAP-T cells) or of classical CAR-T cells by electroporation was described in Example 5.
After expansion, a series of tests were carried out to verify the modified T cells, including the positive rate of ICAP expression, the amplification effect, the ratio of CD4/CD8 positive cells in CD3 positive cells and the ratio of effector memory T(Tem)/central memory T (Tcm) cells in memory T cells (Tm). The expression rate of label polypeptide (M-ICAP expression) on the surface of T cells was determined by flow cytometry using biotin-conjugated anti-His antibody and a PE-conjugated streptavidin secondary antibody. As shown in
This example describes the design and characterization of TCPs used in this application. TCPs used here were bispecific antibodies that can simultaneously recognize target B cell or tumor specific antigens (such as CD19, BCMA and EGFR) and M-ICAP polypeptide (from mesothelin) of the M-ICAP-T cells, so they can be used as an adaptor to control the proliferation or cytotoxicity of M-ICAP-T cells. The TCPs designed and applied in the present working Examples are listed in Table 4.
1. Design and Purification of TCPs
BCMA-TCPs were designed that can simultaneously target BCMA antigen and M-ICAP polypeptide (label derived from mesothelin) for use in cytotoxicity and in vitro efficacy assays described further below. To investigate the effect of different linker formats on the bioactivity and stability of TCPs, three formats of BCMA-TCPs (TCP001-C, TCP002-C and TCP003-C) with different linkers (3×GGGGS linker, hIgG4-Fc, and hIgG4-CH3 respectively) were designed. TCP001-P and TCP001-N were respectively the positive and negative controls in TCP format. At the same time, MC001C and MC001D, targeting BCMA and M-ICAP respectively, were constructed as two positive controls in mAb format.
One CD19-TCP simultaneously targeting CD19 antigen and M-ICAP polypeptide with the 3×G4S linker, named as TCP011-P, was designed for use in the proliferation assay of M-ICAP-T stimulated by CD19 antigen.
One EGFR-TCP simultaneously targeting EGFR antigen and M-ICAP polypeptide with the 3×G4S linker, named as TCP021-P was designed for use in cytotoxicity assay of M-ICAP-T using EGFR expressing solid tumor cell lines as target.
The N terminal M2339VHH sequence targeting M-ICAP polypeptide was identified from phage display with alpaca immune VHH libraries, described above in Example 2 and Example 3. The B029(VHH) sequence targeting BMCA was identified from phage display with alpaca immune VHH libraries, described above in Example 2. The scFv sequence in TCP001-P targeting BMCA was derived from B2121 of CN201580050638. The VHH sequence in TCP001-N targeting GFP was derived from the GFP-specific VHH described by Kubala et al. (M. H. Kubala et al, Protein Sci. 19:2389-2401 (2010) (hereby incorporated by reference in its entirety for description of such VHH and how it is used).
The scFv sequence in TCP011-P targeting CD19 was derived from FMC063, described in Chinese patent application CN201480027401.4 (hereby incorporated by reference in its entirety and for all purposes). The E454 sequence in TCP021-P targeting EGFR was identified from phage display with alpaca immune VHH libraries, described above in Example 2.
The genes of were synthesized and cloned by Genewiz, Inc. All ORF DNAs were cloned into the pcDNA3.4 vector between the BamHI and EcoRI sites. Antibody expression, purification and purity quality control were conducted as described in the publication WO2020176815A2 (hereby incorporated by reference for description of such methods).
2. Affinity Characterization of TCPs
First the binding affinity of the purified BCMA-TCPs against BMCA antigen was assessed by SPR. BCMA-his antigen was coupled onto a CM5 chip (GE Healthcare Life Sciences), and then a variety of anti-BCMA BsAbs were flowed at the flow rate of 10 uL/min with dissociation time of 900s. Binding kinetics were determined with a 1:1 fit model. The data indicated that linker structure might influence the binding affinity, as TCP001-C with a 3×G45 linker had higher binding affinity than TCP002-C and TCP0031-C with larger linkers (Table 5).
The binding bioactivity for mesothelin and BCMA over-expressing cells was evaluated by flow cytometry. Stable cell lines were harvested using 0.25% Trypsin.
About 5E5 cells were collected per sample, and the cells were resuspended in 100 μL/well tested antibodies with His-tag. Then the cells were incubated with anti-His-tag antibody (Genscript, China) and Streptavidin-PE (Biolegend, China). The incubation step of each step was performed at 4° C. in the dark for 1 hr, and then the cells were washed 2× with 200 μL PBS buffer. The washed cells were resuspended in 200 μL PBS buffer and the sample was analyzed by FACS. As shown in
3. The Stability of BCMA-TCPs in Human Plasma In Vitro
TCP001C, TCP002C and TCP003C were incubated in 100% human plasma at 37° C. for up to 21 days, and samples were respectively collected at Day 0, 1, 3, 7, 14, and 21. A 96-well plate was coated with mesothelin antigen, and after plate blocking and washing, collected samples with proper dilution together with serially diluted standard samples were incubated with the plate at 37° C. for 1 hr. Anti-VHH-cocktail-HRP (GenScript, A02016) was used as the detection antibody and the absorbance was read at 450 nm. Finally, the tested samples were analyzed according to the fitted curve of a standard sample group.
As shown in
The binding affinity of TCP011-P for both CD19 and MSLN over-expressing cells (
To verify the rapid activation and amplification of ICAP-T cells with TCP culturing with target cells, PBMC-T cells transfected with M-ICAP (activated by anti-His and anti-CD28) were co-cultured with CD19 positive Daudi lymphoma cells in the presence of TCP011-P or -N, respectively. CD19 positive Daudi lymphoma cells were treated with or without 50 ug/ml mitomycin C for 2 hr. 5×105 PBMC-T cells transfected with M-ICAP cells were counted and cocultured with 5×105 Daudi cells, as well as TCP011-P or -N for 4 days. The effector or target cells were then analyzed for proliferation by flow cytometry.
As shown in
In order to make ICAP-T cells act on BCMA positive tumor cells, it is necessary to have a TCP that can specifically bind to BCMA. The two ends of TCP001-C and -P can specifically bind ICAP-T and BCMA cells at the same time. In order to verify that, combined with specific TCP, ICAP-T can act on BCMA positive tumor cells and have specific cytolysis/killing effect, we compared the cytolysis/killing effect of ICAP-T or CAR-T cells co-cultured with RPMI-8226 or L363 cells (in three different E:T ratio) in the presence of different TCPs.
The cytotoxicity assay of T cells for suspension cell lines was conducted according to the manufacturer's protocol (DELFIA® EuTDA Cytotoxicity Reagents AD0116—PerkinElmer). Briefly, target tumor cells were washed with PBS and fluorescence enhancing ligand and incubated for 15 minutes at 37° C. 50 ul of target cells (5,000 cells) were placed into a V-bottom plate containing bispecific polypeptide that specifically binds to both of the target tumor cells and to the effector cells (that is, the transformed T cells), and 50 ul of effector cells are added with varying cell concentration (E:T=16/8/4:1). After 3.5 hours' incubation, 10 ul of the supernatant were transferred into 100 μL of Europium Solution. After 15 minutes incubation at room temperature, the fluorescence was measured in the time-resolved fluorometer. Specific release (%)=Experimental release (counts)−Spontaneous release (counts)/Maximum release (counts)−Spontaneous release (counts)×100.
IFNγ secretion by the T cells was also assayed. The IFNγ detection was conducted according to the manufacturer's protocol (IFNγ detection kit, VAL104—Novus). Briefly, fresh washing solution, colorant, diluent and standard product were prepared according to instructions. Different concentrations of standards and diluted experimental samples were added into the corresponding wells for 100 ul/well. The reaction well was sealed with sealing tape and incubated at room temperature for 2 hours. After 4 times washing using wash-buffer, 200 uL of enzyme labeled antibody was added into each well for 2 hours incubation at room temperature. After repeating the plate washing operation, 200 uL of previously mixed color reagent is added to each well, and the reaction incubated for 10-30 min in the dark. The color of the solution will change from blue to yellow by adding 50 ul/well termination solution. OD values are recorded with a spectrophotometer in 20 minutes and analyzed in Excel using a selected “four parameter equation” to obtain standard curve using a standard sample group.
As shown in
The two ends of TCP001-C/P, TCP002-C/P and TCP003-C/P bind M-ICAP and BCMA at the same time. To verify and compare the specific cytolysis effect of ICAP-T cells combined with these TCPs on BCMA positive tumor cells, a cytolysis/killing assay of ICAP-T or CAR-T cells co-cultured with RPMI-8226 or L363 cells (E:T=8:1) was performed in the presence of various TCPs. The cytotoxicity and the IFNγ secretion assays of T cells for suspension cell lines are described in Example 9.
As shown in the
In order for ICAP-T cells to act on EGFR positive tumor cells, it is necessary to have a TCP that can specifically bind to EGFR. TCP021-P can combine ICAP and EGFR at its two ends, respectively. To verify that ICAP-T cells combined with this specific TCP can act on EGFR positive tumor cells such as FaDu (human pharyngeal squamous cell carcinoma) and SK-OV3 (human ovarian cancer cells), we compared the killing effect of ICAP-T or CAR-T cells co-cultured with FaDu/SK-OV3 cells in the presence of various TCPs.
The cytotoxicity assay of T cells for adhesion cell lines is conducted using an impedance-based RTCA TP instrument and method (xCELLigence). Target tumor cells are seeded in a resistor-bottomed 96-well plate at 10,000 cells per well within the RTCA TP instrument overnight (more than 16 hours). The bispecific TCP or antibodies are added to the cultured target tumor cells and the cells are further cultured for 30 minutes. Then ICAP-T or CAR-T cells are incubated with target tumor cells at different effector cell:target cell ratios for about 100 hours (the end point depends on the killing efficiency of transformed T cells). During the experiment, the cell index values are closely correlated with tumor cell adherence, such that lower cell attachment indicates higher cytotoxicity, and are collected every 5-10 minutes by the RTCA system. The real-time killing curves are automatically generated by the system software. Specific lysis (%) of each transformed T cell are also calculated using the data of the 48 h point [specific lysis=(cell index of tumor cells alone−cell index of transformed T cells cocultured with tumor cells)/cell index of tumor cells alone].
As shown in
In order for ICAP-T cells to act on B-Cells, it is necessary to have TCP that can specifically bind to CD19. TCP011-P can bind ICAP and CD19 at its two ends, respectively. To verify the effect of ICAP-T cells combined with this specific TCP on CD19 positive B cells, we compared the cytolysis of Daudi cells and the release of IFN-γ when ICAP-T or CAR-T cells were co-cultured with Daudi cells in the presence of various TCPs. The cytotoxicity and the IFNγ secreting detection assay of T cells for suspension cell lines are as described in Example 9.
As shown in the
Due to complicated tumor microenvironment, most CAR-T therapies targeting solid tumors tested clinically at present show little clinical efficacy. To enhance anti-tumor effects of the ICAP-T cell system, M-ICAP-T cells secreting an immune checkpoint inhibitor, for example anti-PD-1, an antagonist of suppression cytokines in tumor, such as anti-TGFβ, or the like, were produced by simultaneously transfecting human naïve T cells with M-ICAP peptide (from MII+III peptide) and a plasmid encoding the secreted immune checkpoint inhibitor.
After 13 days of M-ICAP-T preparation, FACS was used for ICAP expression detection. Results are shown in
ELISA was used to test concentrations of secreting proteins. Supernatants were added to antigen-coated 96-well plates, HRP conjugated anti-VHH and HRP-anti-His were used for anti-PD-1 VHH, anti-PD-L1 VHH and anti-TGFβ scFv detection respectively. Results were shown in
The binding ability of secreted anti-PD-1 VHH was tested indirectly by FACS of T cells expressing PD-1 on their surface. A commercially available anti-PD-1 antibody was used to examine PD-1 expression level on T cells in a competition assay. As
A commercially available TGFβRII-293T-Luc cell line was used to determine blocking activities of secreted anti-TGFβ scFv obtained in Example 13. 5000 TGFβRII-293 cells were seeded and incubated overnight, test samples were added, followed by 5 nM TGFβ, after 6 hr, ONE-GLO was used to read bioluminescence. Results are shown in
The orthotopic tumor model experiment described below utilizes NPSG mice (NOD-Prkdcscid IL2rgtm1/Pnk).
1. Tumor Inoculation, Grouping, Drug Administration and Animal Observations
2. Body Weight; Tumor Measurements
3. Analysis of Anti-PD-1 and TCP001-C Concentration in Whole Blood of Mice
100 μl of peripheral blood was collected once a week for analysis of the count of the anti-PD-1 and TCP001-C concentration in whole blood of mice. 1 μg/ml PD-1 protein was coated on 96-well plates overnight for ELISA binding assay. Diluted samples together with diluted standard samples (8 dilution points from 2 ng/ml) were added to the wells and let stand for 1 hr at 37° C. Then anti VHH-cocktail antibody was added as detection antibody and the assay reagents are added. The absorbance was read at 450 nm. Concentrations were determined by analysis against the standard curve as in Example 9.
A peptide motif (about 20-30 aa in length) fused to the N terminal of the antigen binding domain (scFv or VHH) of a CAR-T cell receptor as a universal ICAP for CAR-T amplification in vitro or in vivo. The following criteria were used for the peptide motif design.
First, the peptide is about 20-30 aa in length. Second, nanobodies specific to this peptide motif with high binding affinity (KD<1 nM) can be obtained. Finally, the nanobodies targeting the peptide successfully induced CAR-T in vitro or in vivo amplification when the peptide was fused to the N terminal of the antigen binding domain (scFv or VHH) of the chimeric antigen receptor of CAR-T cells.
1. Identification and Characterization of One VHH Sequence with High Affinity Towards MSLN.
We identified one VHH nanobody towards MSLN with high affinity using an alpaca immune library. The procedures screening for alpaca immunization, blood collection, library construction, solid panning, ELISA or FACS screening for positive clones, antibody purification and followed antibody characterization by SPR and FACS were as described in Example 2. One positive clone named as anti-MSLN-1444 VHH was obtained.
As shown in Table 6, the affinity of anti-MSLN-1444 VHH towards MSLN His antigen was high with KD of 2.10E-09.
As shown in
2. Identification of BCMA Peptide (BCMA ICAP) which can be Recognized Potently by BCMA Full Length VHH Binders.
One BCMA peptide motif with proper length (˜20 aa) that can be recognized with high affinity by some BCMA candidate binding proteins from a previously prepared immune library with BCMA-hFc antigen and many candidate VHH sequences with various binding properties to full length BCMA was designed. One polypeptide BCMA mut1 from natural BCMA (1-23aa of BCMA ECD domain, Table 7—SEQ ID NO: 17) was selected, and a fusion polypeptide of BCMAmut1 and anti-MSLN-1444 VHH sequence targeting MSLN to be expressed is shown in
Then we filtered out three VHH sequences (#36, #102 and #367 described below) with high affinity to it. The sequence of BCMA mut1 is also shown in Table 7 (SEQ ID NO:18), The three VHH sequences were expressed as a hFc fusion proteins, named as 36(VHH), 102(VHH) and 367(VHH) with the procedure that was described in the patent publication WO2020176815A2 (hereby incorporated by reference in its entirety and for all purposes). The binding affinity of three VHH nanobodies was measured by SPR. As shown in
3. BCMA ICAP can be Used to Specifically Amplify CAR T Cells with BCMA ICAP.
An ICAP-1-23-3GS vector was constructed as illustrated in
4. Anti BCMA Mut1 36# Did not Stimulate Non-Specific Amplification of CAR T Cells with BCMA ICAP.
To test the specificity of stimulation of anti-BCMAmut1 36# to BCMA ICAP CAR-T cells, a MSLN-1444 CAR vector was constructed as shown in
Having shown and described exemplary embodiments of the subject matter contained herein, further adaptations of the methods and systems described herein may be accomplished by appropriate modifications without departing from the scope of the claims. In addition, where methods and steps described above indicate certain events occurring in certain order, it is intended that certain steps do not have to be performed in the order described but in any order as long as the steps allow the embodiments to function for their intended purposes. Therefore, to the extent there are variations of the invention, which are within the spirit of the disclosure or equivalent to the inventions found in the claims, it is the intent that this patent will cover those variations as well. Some such modifications should be apparent to those skilled in the art. For instance, the examples, embodiments, geometrics, materials, dimensions, ratios, steps, and the like discussed above are illustrative. Accordingly, the claims should not be limited to the specific details of structure and operation set forth in the written description and drawings.
Embodiment 1: An immune cell that comprises an expressed immune cell activator polypeptide comprising an intracellular signal transduction domain, a transmembrane domain, and an extracellular label domain, wherein the immune cell secretes one or more polypeptide effector molecules.
Embodiment 2: An immune cell that comprises an expressed immune cell activator polypeptide comprising an intracellular signal transduction domain, a transmembrane domain, and an extracellular chimeric polypeptide comprising a binding domain of a VHH antibody or a single chain variable fragment and a label domain, wherein the immune cell secretes one or more polypeptide effector molecules.
Embodiment 3: The immune cell of embodiment 1 or embodiment 2, wherein the label domain comprises a polypeptide derived from structural membrane protein or fetoprotein.
Embodiment 4: The immune cell of any of embodiments 1-3, wherein the polypeptide effector molecule comprises an antibody or a binding fragment thereof that specifically binds to one or more immunomodulators.
Embodiment 5: The immune cell of embodiment 4, wherein the antibody is a VHH antibody.
Embodiment 6: The immune cell of embodiment 4, wherein the immunomodulator is PD-1, PD-L1, CTLA4, LAG-3, TIM-3, BTLA, CD3, CD27, CD28, CD40, CD160, 2B4, 4-1BB, GITR, OX40, VEGF, VEGFR, TGFβ, TGFβR, HVEM or LIGHT.
Embodiment 7: The immune cell of any one of embodiments 1-6, wherein the label domain specifically binds to a bispecific polypeptide comprising a label-binding domain comprising a single chain polypeptide and a cell surface protein-binding domain comprising a single chain polypeptide that binds to a cell surface receptor of a cell.
Embodiment 8: An immune cell that comprises a nucleic acid vector comprising
Embodiment 9: The immune cell of embodiment 8, which further comprises a second nucleic acid vector comprising
Embodiment 10: The immune cell of embodiment 8, wherein the nucleic acid vector further comprises a polynucleotide encoding an amino acid sequence of one or more secreted polypeptide effector molecules.
Embodiment 11: The immune cell of any one of embodiments 8-10, wherein the immune cell activator polypeptide further comprises a binding domain of a VI-11-1 antibody or a single chain variable fragment.
Embodiment 12: The immune cell of any one of embodiments 8-11, wherein the immune cell activator polypeptide comprises a chimeric polypeptide comprising (i) a binding domain of a VI-11-1 antibody or a single chain variable fragment and (ii) the label domain.
Embodiment 13: The immune cell of embodiment 12, wherein the chimeric polypeptide is branched.
Embodiment 14: The immune cell of any one of embodiments 8-13, wherein the label domain comprises a polypeptide derived from a fetoprotein.
Embodiment 15: The immune cell of any one of embodiments 8-13, wherein the label domain comprises a structural membrane protein.
Embodiment 16: The immune cell of any one of embodiments 8-15, wherein the signal transduction domain comprises a co-stimulation domain and a T Cell Receptor (TCR) signaling domain.
Embodiment 17: The immune cell of embodiment 16, wherein the co-stimulation domain comprises CD28, ICOS, CD27, 4-1BB, OX40 or CD40L.
Embodiment 18: The immune cell of embodiment 16 or embodiment 17, wherein the TCR signaling domain comprises CD3ζ or CD3ε.
Embodiment 19: The immune cell of any one of embodiments 16-18, wherein the signal transduction domain comprises CD28 and CD3ζ.
Embodiment 20: The immune cell of any one of embodiments 8-19, wherein the transmembrane domain comprises a domain that participates in immune co-stimulatory signaling.
Embodiment 21: The immune cell of any one of embodiments 8-20, wherein the transmembrane domain comprises CD28.
Embodiment 22: The immune cell of embodiment 21, wherein the CD28 comprises an ITAM domain.
Embodiment 23: The immune cell of any one of embodiments 8-18 and 20-22, wherein the CD3ε domain comprises the amino acids YMNM.
Embodiment 24: The immune cell according to any one of embodiments 8-23, wherein at least one nucleic acid vector further comprises PiggyBac Transposase.
Embodiment 25: The immune cell according to any one of embodiments 8-23, wherein at least one nucleic acid vector further comprises transposon Inverted Terminal Repeat sequences.
Embodiment 26: The immune cell of any one of embodiments 8-25, wherein the polypeptide effector molecule comprises an antibody or a binding fragment thereof that specifically binds to one or more immunomodulators.
Embodiment 27: The immune cell of embodiment 26, wherein the antibody is a VHH antibody.
Embodiment 28: The immune cell of embodiment 26 or embodiment 27, wherein the immunomodulator is PD-1, PD-L1, CTLA4, LAG-3, TIM-3, BTLA, CD3, CD27, CD28, CD40, CD160, 2B4, 4-1BB, GITR, OX40, VEGF, VEGFR, TGFβ, TGFβR, HVEM or LIGHT.
Embodiment 28: The immune cell of any one of embodiments 8-25, wherein the polypeptide effector molecule comprises a cytokine.
Embodiment 30: The immune cell of embodiment 29, wherein the cytokine is TGF-β, VEGF, TNF-α, CCR5, CCR7, IL-2, IL-7, IL-15 or IL-17.
Embodiment 31: The immune cell of any of embodiments 8-30, which is T cell, tumor infiltrating lymphocyte, cytokine activated killer cell, dendritic cell-cytokine activated killer cell, γδ-T cell, natural killer T cell, or natural killer cell.
Embodiment 32: An immune cell activator polypeptide comprising:
Embodiment 33: The immune cell activator polypeptide of embodiment 32, wherein the signal transduction domain comprises a co-stimulation domain and a T Cell Receptor (TCR) signaling domain.
Embodiment 34: The immune cell activator polypeptide of embodiment 33, wherein the co-stimulation domain comprises CD28, ICOS, CD27, 4-1BB, OX40 or CD40L.
Embodiment 35: The immune cell activator polypeptide of embodiment 33, wherein the TCR signaling domain comprises CD3ζ or CD3ε.
Embodiment 36: The immune cell activator polypeptide of embodiment 33, wherein the signal transduction domain comprises CD28 which is linked at its C-terminal end to the N-terminal end of a CD3ε signaling domain.
Embodiment 37: The immune cell activator polypeptide of embodiment 33, wherein the signal transduction domain comprises a co-stimulation domain 4-1BB which is linked at its C-terminal end to the N-terminal end of a CD3ε signaling domain.
Embodiment 38: The immune cell activator polypeptide of any one of embodiments 32-37, wherein the label domain comprises a polypeptide derived from a fetoprotein.
Embodiment 39: The immune cell activator polypeptide of any one of embodiments 32-37, wherein the label domain comprises a structural membrane protein.
Embodiment 40: The immune cell activator polypeptide of any one of embodiments 32-39, wherein the transmembrane domain comprises a domain that participates in immune co-stimulatory signaling.
Embodiment 41: The immune cell activator polypeptide of any one of embodiments 32-40, wherein the transmembrane domain comprises CD28 or a structural membrane protein.
Embodiment 42: The immune cell activator polypeptide of any one of embodiments 32-41, wherein the CD28 comprises an ITAM domain.
Embodiment 43: The immune cell activator polypeptide of any one of embodiments 32-42, wherein the CD3ε domain comprises amino acids YMNM.
Embodiment 44: A nucleic acid vector comprising
Embodiment 45: The nucleic acid vector according to embodiment 44, which further comprises transposon Inverted Terminal Repeat sequences.
Embodiment 46: A nucleic acid vector comprising:
Embodiment 47: The nucleic acid vector according to embodiment 46, which further comprises transposon Inverted Terminal Repeat sequences.
Embodiment 48: The nucleic acid vector according to embodiment 46 or embodiment 47, wherein the polypeptide effector molecule comprises an antibody or a binding fragment thereof that specifically binds to one or more immunomodulators.
Embodiment 49: The nucleic acid vector according to embodiment 48, wherein the antibody is a VHH antibody.
Embodiment 50: The nucleic acid vector according to embodiment 46 or embodiment 47, wherein the polypeptide effector molecule comprises a cytokine.
Embodiment 51: A bispecific polypeptide comprising:
Embodiment 52: The bispecific polypeptide of embodiment 51, in which the label-binding domain comprises VHH domain of a camelid IgG.
Embodiment 53: The bispecific polypeptide of embodiment 51 or embodiment 52, which comprises about 15-20 amino acids of a CDR3 domain.
Embodiment 54: The bispecific polypeptide of any one of embodiments 51-53, wherein the cell is a lymphocyte.
Embodiment 55: The bispecific polypeptide of embodiment 54, wherein the lymphocyte is a B cell.
Embodiment 56: The bispecific polypeptide of any one of embodiments 51-53, wherein the cell is a tumor cell.
Embodiment 57: The bispecific polypeptide of embodiment 56, wherein the tumor is lymphoma, non-small cell lung cancer, breast cancer, ovarian cancer, liver cancer, or mesothelioma.
Embodiment 58: The bispecific polypeptide of embodiment 56 or 57, wherein the cell surface protein is EGFR.
Embodiment 59: The bispecific polypeptide of embodiment 56 or 57, wherein the cell surface protein is GPC3.
Embodiment 60: The bispecific polypeptide of any one of embodiments 51-57, wherein the cell surface protein-binding domain specifically binds to EGFR protein expressed on the surface of a tumor cell.
Embodiment 61: The bispecific polypeptide of any one of embodiments 51-57, wherein the cell surface protein-binding domain specifically binds to CD19, CD20 or CD22 on the surface of a lymphoma cell.
Embodiment 62: The bispecific polypeptide of any one of embodiments 51-57, which comprises a VI-11-1 antibody.
Embodiment 63: The bispecific polypeptide of any one of embodiments 51-62, which further comprises one or more domains that provide additional biochemical activities or biological functions.
Embodiment 64: The bispecific polypeptide of embodiment 63, wherein the additional biochemical activities or biological functions comprise: specific binding of a fluorophore, extending the half-life of the bispecific polypeptide in vivo, increasing the affinity of the bispecific polypeptide, and modulating an immune response mediated by a Fc domain.
Embodiment 65: The bispecific polypeptide of any of embodiments 51-62, which comprises additional cell surface protein-binding domain(s) comprising a single chain polypeptide domain(s) that bind to different cell surface receptor(s) of the same or different cell.
Embodiment 66: A kit for in situ production of one or more polypeptide effector molecules proximal to a target cell comprising:
Embodiment 67: The kit of embodiment 66, wherein the cell surface protein-binding domain specifically binds to CD19 on a B cell.
Embodiment 68: The kit of embodiment 66 or embodiment 68, wherein the cell surface protein-binding domain specifically binds to EGFR, mesothelin, BCMA, MUC1 or GPC3 on a tumor cell.
Embodiment 69: A method for modulating the immune system environment in the locality of a tumor cell in a subject comprising:
Embodiment 70: The method of embodiment 69, further comprising a step performed between steps a. and b. of measuring the amount of the immune cells in the subject.
Embodiment 71: The method of embodiment 70, in which the amount of the immune cells in the blood of the subject is measured.
Embodiment 72: The method of embodiment 70, in which the amount of the immune cells infiltrating the tumor of the subject is measured.
Embodiment 73: The method of any one of embodiments 69-72, wherein the immune cell is T cell, tumor infiltrating lymphocyte, cytokine activated killer cell, dendritic cell-cytokine activated killer cell, γδ-T cell, natural killer T cell, or natural killer cell.
Embodiment 74: The method of any one of embodiments 69-73, wherein the cell surface protein of the lymphocyte is CD19 of a B cell.
Embodiment 75: The method of any one of embodiments 69-74, wherein the tumor cell is lymphoma cell, mesothelial cell, non-small cell lung cancer cell, ovarian cell, liver cancer, or breast cancer cell.
Embodiment 76: The method of embodiment 75, wherein the cell surface protein is EGFR, mesothelin, BCMA, MUC1 or GPC3.
Embodiment 77: A method for modulating the immune system environment in the locality of a tumor cell in a subject comprising:
Embodiment 78: The method of embodiment 77, wherein the tumor cell is a mesothelial cell that overexpresses mesothelin and PDL1, and the cell surface protein is mesothelin expressed on the surface of a mesothelial cell, and wherein the effector molecule comprises a VI-11-1 domain that specifically binds to PD-1 or to CD40.
Embodiment 79: The method of embodiment 77 or embodiment 78, wherein the tumor cell is a B cell and the cell surface protein is CD19, CD20 or CD22 on the surface of B cells.
Embodiment 80: The method of any one of embodiments 77-79, wherein the immune cell is T cell, tumor infiltrating lymphocyte cytokine activated killer cell, dendritic cell-cytokine activated killer cell, γδ-T cell, natural killer T cell, or natural killer cell.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2020/001079 | 12/28/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62954448 | Dec 2019 | US |
