Switchable CAR-T Therapy for Treating Human Cancers

Abstract

The present invention provides a method for treating CD19-positive malignancies in human subjects using an appropriate dose of switchable CAR-T cells and complementary switch molecules.

Description

[Technical field]

[0001] REFERENCE TO RELATED APPLICATIONS This patent application claims priority to U.S. Provisional Patent Application No. 63 / 286,868, filed December 7, 2021, currently pending. The entire disclosure of the priority application is incorporated by reference in its entirety into this disclosure for all purposes. [Background technology]

[0002] Despite the enormous clinical benefits, adverse events associated with CAR-T cell therapy remain a challenge. The most frequent adverse events are cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome due to the inability to regulate the activity levels of current CAR-T cell products after administration to patients. Other challenges include on-target toxicity, off-tumor toxicity, and disease relapse mediated by antigen deficiency.

[0003] To address these challenges, a “switchable” CAR-T (sCAR-T) platform has been developed in which the activity of sCAR-T cells is controlled by an antibody-based switch. The switch targets tumor antigens, and the sCAR recognizes a unique peptide embedded in the switch. The switch forms a bridge between the sCAR-T cells and tumor cells, activating the sCAR-T cells to induce tumor cell killing. Combining the switch cells with sCAR-T cells completely eliminates tumors in xenograft and allograft models, but each is designed to be inactive alone. The short half-life of the switch allows for rapid tuning of the activity of sCAR cells through switch administration. Furthermore, by swapping in a different switch, sCAR-T cells can be redirected to other tumor targets using a modular approach. Cyclic on / off stimulation of sCAR-T cells has been shown to improve sCAR-T cell memory and persistence.

[0004] There is a strong need in the art for practical application of the sCAR-T platform in the treatment of human subjects, and the present invention is directed to addressing this and other outstanding needs. Summary of the Invention [Means for solving the problem]

[0005] In one aspect, the invention provides a method of treating a CD19-positive malignancy, inhibiting tumor growth, and / or promoting tumor regression in a human subject. The method comprises administering to the subject (a) a chimeric antigen receptor T cell switch molecule (CAR-T switch) comprising an anti-CD19 Fab antibody comprising light and heavy chain variable region sequences shown as SEQ ID NO: 2 and 3, respectively, and (b) a complementary CAR-T cell comprising a CAR sequence shown as SEQ ID NO: 6, thereby treating the B cell malignancy, inhibiting tumor growth, and / or promoting tumor regression in the subject. In some embodiments, the anti-CD19 Fab antibody used comprises light and heavy chain sequences shown as SEQ ID NO: 15 and 16, respectively. In some embodiments, the subject is administered a single dose of CAR-T cells at the start of treatment and multiple doses of the CAR-T switch during the course of treatment. In some of these embodiments, the subject is infused with a single dose of CAR-T cells, followed by one or more infusion cycles of the CAR-T switch. Each infusion cycle includes an "on" phase in which the CAR-T switch is infused daily for about 5 to about 9 days, and an "off" phase in which no CAR-T is administered for about 14 to about 28 days.

[0006] In various embodiments, the dose of CAR-T cells administered to a subject is about 60×10 cells. 6 pieces, approximately 80×10 6 pieces, approximately 100×10 6 pieces, approximately 120×10 6 pieces, approximately 140×10 6 pieces, approximately 160×10 6 pieces, approximately 180×10 6 pieces, approximately 200×10 6 pieces, approximately 300×10 6pieces, approximately 400×10 6 pieces, approximately 500×10 6 pieces, approximately 600×10 6 pieces, approximately 700×10 6 pieces, approximately 800×10 6 pieces, approximately 900×10 6 pieces, approximately 1000×10 6 In some embodiments, the dose of CAR-T cells administered to a subject is about 0.35×10 cells or more. 8 From about 14 x 10 8 In some of these embodiments, the dose of CAR-T cells administered to a subject is about 1.4 x 10 cells. 8 From about 7 x 10 8 In one embodiment, the dose of CAR-T cells administered to a subject is about 1.4×10 cells. 8 It is one piece.

[0007] In some methods of the invention, the dose of the CAR-T switch administered to the subject is about 0.01 mg to about 0.1 mg per kg of body weight. In various embodiments, the dose of the CAR-T switch administered to the subject can be about 0.01 mg, about 0.025 mg, about 0.03 mg, about 0.04 mg, about 0.045 mg, about 0.05 mg, about 0.055 mg, about 0.06 mg, about 0.065 mg, about 0.070 mg, about 0.075 mg, about 0.085 mg, or about 0.095 mg per kg of body weight. In some embodiments, the dose of the CAR-T switch administered to the subject is about 0.045 mg to about 0.075 mg per kg of body weight. In one embodiment, the dose of the CAR-T switch administered to the subject is about 0.06 mg per kg of body weight.

[0008] In some methods of the invention, the subject's CD19-positive malignancy being treated is a CD19-positive B-cell cancer. In some methods, the subject's CD19-positive malignancy being treated is a relapsed / refractory B-cell malignancy.

[0009] In a related aspect, the invention provides a method of treating a CD19-positive relapsed / refractory B-cell malignancy in a human subject, inhibiting tumor growth and / or promoting tumor regression, comprising administering to a human subject at least about 0.35×10 CAR-T cells. 8 pieces~approx. 7×10 8 (b) administering to the subject a single dose of CAR-T cells comprising the CAR sequence set forth in SEQ ID NO: 6; and (b) administering to the subject a CAR-T switch molecule that is an engineered anti-CD19 Fab antibody comprising light and heavy chain sequences set forth as SEQ ID NOs: 15 and 16, respectively, during one or more infusion cycles, each of which comprises (i) an "on" phase of about 5 to about 9 days and (ii) an "off" phase of about 14 to about 28 days, wherein a daily dose of CAR-T infused to the subject during the "on" phase is used of about 0.045 mg to about 0.075 mg per kg of body weight. Preferably, the CAR-T cells administered are autologous to the subject. In some embodiments, the infusion dose of CAR-T cells is about 1.4×10 cells. 8 The daily dose of the CAR-T switch infused during the "ON" phase is about 0.06 mg / kg body weight. In some of these embodiments, the "ON" phase is about 7 days and the "OFF" phase is about 21 days. In various embodiments, the number of cycles of CAR-T switch infusion can be 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

[0010] In some methods, the subject is a subject treated with lymphodepleting preconditioning before the CAR-T cells and the switch molecule are administered. In some of these embodiments, lymphodepleting preconditioning of the subject can be achieved by chemotherapy with cyclophosphamide and fludarabine. In various embodiments, the CD19-positive relapsed / refractory B-cell malignancy treated in the subject can be, for example, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), hairy cell leukemia (HCL), primary intraocular lymphoma, Burkitt's lymphoma, or Waldenstrom's macroglobulinemia.

[0011] A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification and claims. [Brief description of the drawings]

[0012] [Figure 1]

[0033] Figure 1 shows an in vivo study using the MABEL approach to determine first-in-human (FIH) dose. [Diagram 2] 1 shows a simulation of the human equivalent dose of the CAR-T switch molecule SWI019. [Diagram 3] CT scans showing the efficacy of treatment in patients are shown. [Figure 4] 1 shows resolution of toxicity in treated patients. [Diagram 5] Schematic of the Phase I dose escalation study of sCAR-T therapy. A. Phase I dose escalation study design. B. Overview of treatment schedule. [Figure 6] Dosage recommendations based on the BAYDE model and clinical utility index are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The present invention is based in part on studies conducted by the inventors to determine appropriate doses of switchable CAR-T therapy in human patients, as described in detail below. In particular, through a dose-escalation Phase I human clinical trial, the inventors were able to determine appropriate doses of CD19-targeting sCAR-T platform switches and corresponding CAR-T cells with specificity in the treatment of several CD19-positive malignancies.

[0014] Thus, the present invention provides methods and dosing regimens for treating human patients with various cancers, e.g., CD19-expressing tumors, using the sCAR-T platform described in this disclosure. Unless otherwise indicated, the present invention is directed to, for example, the method and apparatus described in Methods in Enzymology, Volume 289: Solid-Phase Peptide Synthesis, JN Abelson, MI Simon, GB Fields (editors), Academic Press, 1st edition (1997) (ISBN-13: 978-0121821906), U.S. Pat. Nos. 4,965,343 and 5,849,954, Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (1982), Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (1989), Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1986), or Methods in Enzymology: Guide to Molecular Cloning Techniques Vol. 152, edited by SLBerger and ARKimmerl, Academic Press Inc., San Diego, USA (1987), Current Protocols in Protein Science (CPPS) (edited by John E. Coligan, et.al., John Wiley and Sons, Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et.al., John Wiley and Sons, Inc.) and Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss, 5th edition (2005), Animal Cell Culture Methods (Methods in Cell Biology, Vol. 57, Jennie P. Mather and David Barnes, eds., Academic Press, 1st edition, 1998).

[0015] The following sections further describe basic principles for carrying out the compositions and methods of the present invention.

[0016] I. Definition Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For general definitions of many of the terms used in this invention, see Academic Press Dictionary of Science and Technology, Morris (ed.), Academic Press (1999). st ed., 1992), Oxford Dictionary of Biochemistry and Molecular Biology, Smith et al. (ed.), Oxford University Press (revised edition, 2000), Encyclopaedic Dictionary of Chemistry, Kumar (ed.), Anmol Publications Pvt. Ltd. (2002), Dictionary of Microbiology and Molecular Biology, Singleton et al. (ed.), John Wiley & Sons (3 rd ed., 2002), Dictionary of Chemistry, Hunt (ed.), Routledge (1 sted., 1999), Dictionary of Pharmaceutical Medicine, Nahler (ed.), Springer-Verlag Telos (1994), Dictionary of Organic Chemistry, Kumar and Anandand (eds.), Anmol Publications Pvt. Ltd. (2002), and A Dictionary of Biology (Oxford Paperback Reference), Martin and Hine (eds.), Oxford University Press (4 th Those skilled in the art will understand by reference to the following text (ed., 2000). Some of the terms that apply specifically to the present invention are explained in more detail below.

[0017] It is noted that, as used in this disclosure and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as a precondition for using exclusive language, such as "sole," "only," and the like, in connection with the recitation of claim elements or the use of a "negative" limitation.

[0018] The term "antibody" generally refers to one or more polypeptide chains that exhibit strong monovalent, bivalent or polyvalent binding to a given antigen, one or more epitopes. Unless otherwise specified, antibodies or antibody fragments as used herein include some immunoglobulin molecules or variant antibodies that do not have specific antigen binding activity, such as catalytic antibodies. Immunoglobulins can have sequences from any vertebrate species. They can be produced using any suitable technique, for example, hybridoma technology, ribosome display, phage display, gene shuffling libraries, semi-synthetic or fully synthetic libraries, or combinations thereof. Unless otherwise specified, the term "antibody" as used herein includes intact antibodies, antibody fragments, and other designer antibodies as described below or known in the art (see, for example, Serafini, J Nucl. Med. 34:533-6, 1993).

[0019] An intact "antibody" typically comprises at least two heavy (H) chains (about 50-70 kD) and two light (L) chains (about 25 kD) interconnected by disulfide bonds. Recognized immunoglobulin genes that code for antibody chains include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively.

[0020] Each heavy chain of an antibody consists of a heavy chain variable region (V H ) and the heavy chain constant region. The heavy chain constant region of most IgG isotypes (subclasses) is C H1 , C H2 and C H3 Some IgG isotypes, such as IgM and IgE, contain a fourth constant region domain, C H4 Each light chain is composed of a light chain variable region (V L) and the light chain constant region. The light chain constant region consists of one domain, C L The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibody mediate the binding of the immunoglobulin to various cells of the immune system and to host tissues and factors, including the first component (Clq) of the classical complement system.

[0021] V of antibody H Area and V L The V domains are further subdivided into hypervariable regions, also called complementarity determining regions (CDRs), which are interspersed with more conserved framework regions (FRs). H and V L is composed of three CDRs and four FRs, which are arranged from the amino terminus to the carboxyl terminus in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The positions and numbering system of the CDR and FR regions are defined, for example, by Kabat et al., Sequences of Proteins of Immunological Interest, USDepartment of Health and Human Services, USGovernment Printing Office (1987 and 1991).

[0022] As used in this disclosure, an antibody fragment (or an antigen-binding fragment of an antibody) refers to at least one V-binding fragment from an antibody, given other non-immunoglobulins. H , V L Or C H This refers to any protein or polypeptide that contains an immunoglobulin domain or a component that is not of antibody origin. Such molecules include (i) Immunoglobulin C H F of a receptor or binding protein containing a receptor component having all or part of the domain c fusion protein, (ii) V H And / or V L(iii) a binding protein in which the domain is attached to another molecular scaffold; or (iii) immunoglobulin V H and / or V L and / or C H This includes, but is not limited to, molecules in which domains are combined and / or assembled in a manner not normally found in naturally occurring antibodies or antibody fragments.

[0023] "Humanized" forms of non-human (e.g., rodent (e.g., mouse or rabbit)) immunoglobulins are immunoglobulins that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized immunoglobulins are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody), such as mouse, rat, hamster, rabbit, chicken, cow or a non-human primate, having the desired specificity, affinity, and capacity. In some cases, Fv framework region (FR) residues of the human immunoglobulin are also replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or the donor antibody. These changes are made to further improve antibody performance. In general, humanized immunoglobulins will comprise substantially all of at least one, and typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of a non-human immunoglobulin and all or substantially all of the FR regions being those of a human immunoglobulin sequence. A humanized immunoglobulin may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al. (1986) Nature 321:522-525, Riechmann et al. (1988) Nature 332:323-329, and Presta (1992) Curr. Op. Struct. Biol. 2:593-596.

[0024] The term "human immunoglobulin" as used in this disclosure is intended to include immunoglobulins having variable and constant regions derived from human germline immunoglobulin sequences. The human immunoglobulins of the invention can include, for example, in the CDRs, particularly CDR3, 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). However, the term "human immunoglobulin" as used in this disclosure is not intended to include immunoglobulins in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0025] Lymphocyte depletion (LD) chemotherapy or lymphocyte depletion preconditioning refers to a treatment administered to a subject prior to CAR-T cell infusion to deplete endogenous T cells (and Tregs) so that they will not compete / suppress the infused CAR-T cells and attempt to expand / grow the infused CAR-T cells. An example of LD preconditioning is administering fludarabine + cyclophosphamide (FluCy) to a subject.

[0026] The terms "specific binding" or "specifically binds" or "specific for" refer to binding of a binding moiety to a binding target, such as binding of an immunoglobulin or small molecule drug to a target molecule or antigen (e.g., an epitope on a specific polypeptide, peptide or other target (e.g., a glycoprotein target)) that is measurably different from a non-specific interaction (e.g., a non-specific interaction can be binding to bovine serum albumin or casein). Specific binding can be measured, for example, by determining binding of a binding moiety (e.g., a small molecule drug) or immunoglobulin to a target molecule compared to binding to a control molecule. For example, specific binding can be determined by competition with a control molecule that is similar to the target (e.g., unlabeled target in excess). In this case, specific binding is indicated if binding of the labeled target to the probe is competitively inhibited by excess unlabeled target.

[0027] The terms "specific binding" or "specifically binds" or "specific" for a particular target molecule or an epitope on a particular target molecule include, for example, a K d or at least about 200 nM, or at least about 150 nM, or at least about 100 nM, or at least about 60 nM, or at least about 50 nM, or at least about 40 nM, or at least about 30 nM, or at least about 20 nM, or at least about 10 nM, or at least about 8 nM, or at least about 6 nM, or at least about 4 nM, or at least about 2 nM, or at least about 1 nM, or more. In certain instances, the term "specific binding" refers to binding by a binding moiety to a particular target molecule or epitope on a target molecule and to the extent that it does not bind to any other molecules or epitopes.

[0028] In this disclosure, the term "fusion" is used to refer to the combination of amino acid sequences of different origins into one polypeptide chain by combining in-frame the nucleotide sequences encoding them. In addition to fusion to one of the termini of the polypeptide chain, the term "fusion" explicitly includes internal fusions, i.e., the insertion of a sequence of different origins into a polypeptide chain. In this disclosure, the term "fusion" is used to refer to the combination of amino acid sequences of different origins.

[0029] The term "epitope" includes any molecular determinant capable of specific binding to an immunoglobulin. In some embodiments, epitopic determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and in some embodiments can have specific three dimensional structural characteristics and / or specific charge characteristics. An epitope is the region of an antigen to which an immunoglobulin binds. A "binding region" is the region on a binding target to which a binding molecule binds.

[0030] The term "target" or "binding target" is used in the broadest sense and specifically includes polypeptides, but also includes, but is not limited to, nucleic acids, carbohydrates, lipids, cells, and other molecules that occur in nature and may or may not have a biological function. In certain embodiments, the term "target" refers to a cell surface molecule on a target cell, e.g., a tumor cell.

[0031] The term "antigen" refers to a substance or fragment thereof that is capable of binding to an immunoglobulin or eliciting a cellular immune response. Immunogen refers to an antigen that is capable of eliciting an immune response in an organism, particularly an animal, more particularly an immune response in a mammal, including a human. The term "antigen" includes regions known as antigenic determinants or epitopes, as defined above.

[0032] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a presequence or secretory leader DNA is operably linked to a polypeptide DNA if it is expressed as a preprotein involved in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; and a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of secretory leaders, contiguous and in reading frame, although enhancers need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0033] "Percent (%) amino acid sequence identity" to a peptide or polypeptide sequence, i.e., a scFV antibody polypeptide sequence or a peptide derived from GCN4 as specified in this disclosure, is defined as the percentage of amino acid residues in the candidate sequence that are identical to amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conventional substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment can be determined by one of skill in the art, including the algorithms necessary to achieve maximal alignment over the entire length of the sequences being compared. Two sequences are "substantially identical" if, when compared and aligned over a comparison window, i.e., a designated region as determined using one of the known sequence comparison algorithms, or by manual alignment and visual inspection, the two sequences have a specified percentage of identical amino acid residues or nucleotides (i.e., 60% identity over a designated region, or, if not specified, the entire sequence), which may be 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identity).

[0034] "Treatment" or "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to stop or slow (alleviate) the progression of the disease state or disorder of interest. Subjects in need of treatment include those already suffering from the disorder, as well as those susceptible to the disorder and those in whom the progression of the disorder is to be stopped. For example, a subject or mammal is successfully "treated" for cancer if, after the subject or mammal is subjected to the treatment of the present invention, the subject shows an observable and / or measurable reduction or disappearance in one or more of the following: a reduction in the number of cancer cells or the disappearance of cancer cells; a reduction in tumor size; inhibition (i.e., delayed to a predetermined extent, preferably stopped) of cancer cell invasion into surrounding organs, including the spread of cancer to soft tissue and bone; inhibition (i.e., delayed to a predetermined extent, preferably stopped) of tumor metastasis; inhibition of tumor growth to a predetermined extent; and / or a predetermined degree of alleviation of one or more symptoms associated with a particular cancer; a reduction in morbidity and / or mortality; and an improvement in quality of life issues.

[0035] The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, conservatively modified variants refer to nucleic acids that code for the same or essentially identical amino acid sequences, or essentially identical sequences when the nucleic acid does not code for an amino acid sequence. Because the genetic code is degenerate, any protein is encoded by a large number of functionally identical nucleic acids. For example, the codons GCA, GCC, GCG, and GCU all code for the amino acid alanine. Thus, at every position where alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are referred to as "silent variations," and are a type of conservatively modified variation. Every nucleic acid sequence of the present disclosure that encodes a polypeptide also describes every possible silent variation of the nucleic acid. Those skilled in the art will understand that each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be altered to produce a functionally identical molecule. Thus, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each sequence described.

[0036] In the context of polypeptide sequences, "conservatively modified variants" refers to variants with conservative amino acid substitutions, i.e., variants in which an amino acid residue is replaced with another amino acid residue having a side chain with a similar charge. The art has defined families of amino acid residues with similarly charged side chains. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), polar uncharged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0037] The term "contacting" has its standard meaning and refers to combining two or more agents (e.g., polypeptides or phages), combining agents with cells, or combining two populations of different cells. Contacting can occur in vitro, such as by mixing an antibody with cells, or by mixing a population of antibodies with a population of cells in a test tube or growth medium. Contacting can also occur within a cell, i.e., in situ, such as by co-expression in the cell of a recombinant polynucleotide encoding the two polypeptides, causing two polypeptides to be contacted within the cell, or by contacting two polypeptides in a cell lysate. Contacting can also occur in vivo within a subject, such as by administering an agent to the subject to deliver the agent to a target cell.

[0038] The term "subject" often refers to both humans and non-human animals, particularly non-human mammals. Unless otherwise indicated, when referring to the disclosed therapeutic methods, the term preferentially refers to human patients.

[0039] Artificial T cell receptors (also known as chimeric T cell receptors, chimeric immune receptors, chimeric antigen receptors (CARs) or T-bodies) are engineered receptors that engineer arbitrary specificities into immune effector cells. Typically, such receptors are used to engineer monoclonal antibody specificities into T cells, and the transfer of the coding sequences is facilitated by retroviral or lentiviral vectors or transposons. CAR engineered T cells (referred to in this disclosure as CAR-T cells or CAR +CARs (also abbreviated as T cells) are genetically engineered T cells equipped with chimeric receptors whose extracellular recognition site is composed of an antibody-derived recognition domain and whose intracellular region is derived from one or more lymphocyte stimulatory moieties. The architecture of prototype CARs is modular and designed to accommodate various functional domains, allowing the selection of T cell specificity and control of activation. The preferred antibody-derived recognition site is a single chain variable fragment (scFv), which combines the specificity and binding residues of both the heavy and light chain variable regions of a monoclonal antibody. The most common lymphocyte activation moieties include T cell co-stimulatory (e.g., CD28 and / or 4-1BB) domains in tandem with a T cell triggering moiety (e.g., CD3zeta). Providing such chimeric receptors on effector lymphocytes (e.g., T cells or natural killer cells) redirects the genetically engineered cells to any desired target antigen with a defined specificity in an HLA-free manner. CAR constructs are introduced ex vivo into T cells taken from peripheral lymphocytes of a particular patient using retroviral or lentiviral vectors or transposons. Following infusion of the resulting CAR-modified T cells into the patient, the T cells migrate to the target site and, upon interaction with target cells or tissues, undergo activation and exert their defined effector functions. Therapeutic targets of the CAR approach include cancer and HIV-infected cells or autoimmune effector cells.

[0040] A "vector" is a replicon, such as a plasmid, phage, cosmid, or the like, to which another polynucleotide segment can be attached so as to bring about the replication of the attached segment. Vectors capable of directing the expression of genes encoding one or more targeted polypeptides are termed "expression vectors."

[0041] As used in this disclosure, sCAR-T platform refers to a CAR-T switch molecule and a complementary CAR-T cell (also referred to as sCAR-T cell as used in this disclosure). The CAR-T switch molecule (CAR-T switch) includes a targeting moiety (e.g., an antibody or an antigen-binding fragment thereof) that can specifically bind to a target molecule on the surface of a target cell (e.g., a tumor cell). The CAR-T switch can also bind to the CAR of the complementary CAR-T cell. Typically, the extracellular domain of the CAR of the CAR-T cell includes an antibody moiety (e.g., scFv) that specifically recognizes the CAR-ID domain (e.g., a peptide or small molecule) of the CAR-T switch.

[0042] II. Switchable CAR-T Platforms for Treating Human Patients The present invention provides sCAR-T therapy for treating human patients suffering from various cancers, tumors and malignancies. In some embodiments, the method is intended to treat or ameliorate symptoms of a CD-19 positive malignancy in a patient. In related embodiments, the novel therapy of the present invention is directed to promoting tumor regression and / or inhibiting tumor growth in a subject. In some embodiments, a subject treated with the therapy of the present invention is preconditioned with lymphocyte depleting chemotherapy (also known as LD preconditioning). This LD preconditioning is a mandatory step before the subject can actually be infused with CAR-T cells. This creates a "favorable" environment for the expansion and survival of CAR T cells in vivo, and is likely created by eliminating regulatory T cells. LD preconditioning can improve tumor immunogenicity and improve disease control. LD preconditioning has been shown to promote homeostatic proliferation of adoptively transferred T cells by eliminating competition with wild-type T cells and increasing survival / growth-promoting cytokines, namely interleukin (IL)-7 and IL-15. Pre-treatment lymphodepletion preconditioning can be easily performed by methods well known in the art, such as the cyclophosphamide and fludarabine conditioning chemotherapy exemplified in this disclosure. See, e.g., Paplham et al., Leuk Res Rep. 3:28-31, 2014; Bot et al., Blood 126:4426, 2015; Hirayama et al., Blood 133:1876-1887, 2019; Hay and Turtle, Drugs 77:237-245, 2017 and Yakoub-Agha et al., Haematologica. 105:297-316, 2020.

[0043] In many cases, the subject being treated (which may have been administered LD chemotherapy) is administered a switchable CAR-T cell platform (sCAR-T) designed for the treatment of the particular cancer the subject is suffering from, e.g., a CD19-positive B-cell malignancy. The switchable CAR-T cell platform includes (a) a CAR-T switch (also referred to in this disclosure as a CAR-T switch molecule (including a CAR-T switch polypeptide and a CAR-T switch compound)) that can bind to the CAR of the CAR-T cell and specifically targets a cell surface molecule on a tumor cell, and (b) a complementary CAR-T cell that includes a CAR to which the switch can bind. When treating a human subject, it is preferred that the administered CAR-T switch and complementary CAR-T cell are human or humanized. For example, the CAR can be a humanized polypeptide and the T cell expressing the CAR can be a human cell. In some of these embodiments, the T cell expressing the humanized CAR is an autologous T cell isolated from the particular human subject being treated. In many cases, administration can be performed according to standard protocols for immunotherapy, or as outlined in more detail in this disclosure. In some preferred embodiments, the CAR-T switched and complementary CAR-T cells are administered to the subject by infusion. In some embodiments, the sCAR-T treatment methods described in this disclosure can be used in combination with other known therapies or therapeutic agents used to treat cancer, such as chemotherapy, hormone therapy, radiation therapy, or surgery.

[0044] In some methods, one or more CAR-T switches can be administered to a subject to be treated along with complementary CAR-T cells, which target different surface molecules on tumor cells. In some preferred embodiments, different CAR-T switch molecules contain the same CAR-ID domain, which allows different switches to interact with the same complementary CAR-T cells. Such treatments are particularly useful for tumors with heterogeneity. For example, a patient suffering from leukemia or lymphoma can be treated with a pharmaceutical composition comprising (a) both a CD19-targeting CATR-T switch and a CD20 or CD22-targeting switch, and (b) complementary CAR-T cells. In these embodiments, the CAR-T switches can be administered sequentially or simultaneously. A second switch targeting a second cell surface molecule on the target cells can be administered following downregulation of a first cell surface molecule on the target cells targeted by the first switch.

[0045] In a related embodiment, the method of the invention can be used broadly for the transplantation and expansion of CAR-T cells in a subject. Typically, the subject is a subject suffering from a disease or condition (e.g., cancer) that the CAR-T cells are intended to treat. In the method, the subject is administered (a) a CAR-T switch that includes (i) a chimeric antigen receptor-interacting domain (CAR-ID) and (ii) a targeting moiety specific for a molecule (e.g., a tumor cell surface molecule) that manifests the disease or condition suffered by the subject, and (b) a complementary CAR-T cell having a single chain variable fragment (scFv), where the scFv specifically binds to the CAR-ID at the extracellular domain of the CAR of the complementary CAR-T cell.

[0046] Typically, the switchable CAR-T cell platform used in the methods of the invention comprises one or more CAR-T switch molecules and one or more complementary CAR-T cells. In some embodiments, the one or more switches administered and the CAR-T cells are complementary to each other. Typically, the CAR-T switch administered to the subject comprises a chimeric antigen receptor interacting domain (CAR-ID) and a targeting domain or targeting moiety. The CAR-ID specifically binds to the extracellular domain of the CAR on the complementary CAR-T cell. The CAR-ID of the CAR-T switch is any entity that can be fused, conjugated, or otherwise attached to a targeting moiety described in this disclosure (e.g., an anti-CD19 antibody or an antigen-binding portion thereof) such that the CAR-ID can be bound by the CAR of the CAR-T cell. For example, the CAR-ID can be a CAR-binding protein, a CAR-binding peptide, or a CAR-binding small molecule. In some preferred embodiments, the CAR-ID comprises a yeast transcription factor GCN4 peptide or a derivative or homolog thereof. See, e.g., Hinnebusch and Fink, Proc Natl Acad Sci USA 80:5374-8, 1983; Arndt et al., Proc Natl Acad Sci USA 83:8516-20, 1986; WO 2015 / 057834 and WO 2015 / 057852. In some of these embodiments, the yeast transcription factor GCN4 peptide comprises a GCN4 (7P14P) peptide sequence or epitope as described in Berger et al. FEBS Letters 450:149-153, 199 and Zahnd, C., et al., J. Biol. Chem. 279:18870-18877, 2004. As an example, a peptide derived from GCN4 within a CAR-ID can include the sequence NYHLENEVARLKKL (SEQ ID NO: 1) or the sequence RMKQLEPKVEELLPKNYHLENEVARLKKLVGER (SEQ ID NO: 13).

[0047] The targeting moiety of the CAR-T switch used can bind to any target molecule present on the surface of the target tumor cell, for example, CD19 as exemplified in this disclosure. The targeting molecule can preferably include an antigen. In various embodiments, the targeting molecule can be a protein, a lipid moiety, a glycoprotein, a glycolipid, a carbohydrate, a polysaccharide, a nucleic acid, an MHC-binding peptide, or a combination thereof. In some preferred embodiments, the targeting moiety is a targeting polypeptide, such as a targeting antibody or an antigen-binding fragment thereof (e.g., a Fab as exemplified in this disclosure). The targeting antibody can be a human antibody, a fully human antibody, a humanized antibody, a human genetically engineered antibody, a non-human antibody, and / or a chimeric antibody. In some embodiments, the non-human antibody used in the CAR-T switch can be humanized to reduce immunogenicity in humans while retaining the specificity and affinity of the parent non-human antibody. When treating a human patient, the targeting antibody is preferably a humanized or human antibody. In various embodiments, the targeting antibodies can specifically bind to different target molecules on tumor cells, such as CD19, Her2, CLL1, CD33, CD123, EGFR, EGFRvIII, CD20, CD22, CS1, BCMA, CEA, or fragments thereof.

[0048] In some preferred embodiments, the targeting moiety of the CAR-T switch is an antibody or antigen-binding fragment comprising a humanized heavy chain sequence and / or a humanized light chain sequence. Some specific examples of tumor-targeting CAR-T switches that can be used and / or humanized for use in the methods of the invention are described, for example, in PCT / US2017 / 057460, PCT / US2014 / 060713, PCT / US2014 / 060684, PCT / US2016 / 024524, PCT / US2016 / 027997, and PCT / US2016 / 027990.

[0049] In some embodiments, the CAR-ID is fused to the targeting moiety. In some of these embodiments, a structural component of the CAR-ID (e.g., a peptide terminus) is tethered or linked to the terminus of a polypeptide targeting moiety (e.g., a humanized anti-CD19 antibody or antigen-binding fragment thereof). In some embodiments, the CAR-ID is fused to the targeting moiety via a linker. In some embodiments, the CAR-ID is site-specifically attached to the targeting moiety. Site-specific attachment may involve attaching the CAR-ID to a predefined site on the targeting moiety, for example, to the N-terminus of the light chain of the targeting antibody, such as in the exemplified switch molecules of this disclosure. In some embodiments, site-specific attachment may involve attaching the CAR-ID to a non-natural amino acid of the targeting moiety. As a specific example, the CAR-T switch used in the methods of the invention is the CD19 targeting molecule SWI019. This is a modified Fab antibody that contains the GCN4-derived peptide NYHLENEVARLKKL (SEQ ID NO: 1) fused to the N-terminus of the light chain of a humanized anti-CD19 Fab molecule via a GGGGS (SEQ ID NO: 14) linker. The resulting GCN peptide-fused light chain variable region sequence of the Fab molecule is shown in SEQ ID NO: 2. In addition to this light chain sequence, the SWI019 switch (modified Fab molecule) also has a heavy chain variable region sequence shown in SEQ ID NO: 3. The light and heavy chain sequences of SWI019, including the constant region, are shown in SEQ ID NOs: 15 and 16, respectively. More detailed information on the structure of this CAR-T switch is described, for example, in US Pat. No. 11,174,306 and Rodgers DT, et al., PNAS, 2016; 113(4): E459-68.

[0050] Along with the CAR-T switch, complementary CAR-T cells are also administered to the subject for the treatment of cancer. Typically, the CAR-T cell comprises a chimeric antigen receptor (CAR) that includes an extracellular domain, a transmembrane domain, and an intracellular signaling domain. The extracellular domain can specifically bind to the CAR-ID of the CAR-T switch used (e.g., GCN4, Flag, K4, or E4 peptide, or a small molecule such as FITC). In some preferred embodiments, the extracellular domain of the CAR comprises an antibody or an antibody fragment (e.g., scFv) that binds to the CAR-ID of the switch. The antibody may be a human antibody, a fully human antibody, a humanized antibody, a human genetically engineered antibody, a non-human antibody, and / or a chimeric antibody. To treat a human patient, it is preferred that the antibody of the CAR of the CAR-T cell is a human antibody or a humanized antibody. Several known protein transmembrane domains can be used in the CAR of the CAR-T cell. In some embodiments, the transmembrane domain can be the transmembrane domain of CD8 or CD28. In many cases, the intracellular signaling domain can comprise a signaling domain, such as CD3ζ, FcR-γ, Syk-PT, and a co-signaling domain, such as CD28, 4-1BB, CD134, etc. In some embodiments, the intracellular signaling domain of the CAR can comprise (a) a CD3ζ domain, as well as (b) a CD28 domain, a 4-1BB domain, or both a CD28 domain and a 4-1BB domain. In some embodiments, a hinge region is present in the CAR that connects the extracellular domain and the transmembrane domain.

[0051] Depending on the CAR-ID of the CAR-T switch used, various CAR-ID binding moieties can be present on the extracellular domain of the complementary CAR-T cell. In some embodiments, the extracellular domain of the CAR can comprise an antibody or antibody fragment that recognizes yeast transcription factor GCN4 or a fragment thereof. See, for example, Rodgers et al., Proc Natl Acad Sci USA 113:E459-E468, 2016. In some embodiments, the extracellular domain of the CAR of the CAR-T cell can comprise an anti-fluorescein isothiocyanate (FITC) antibody or a FITC-binding portion thereof. The anti-FITC antibody can be an anti-FITC scFv. In some embodiments, the extracellular domain of the CAR of the CAR-T cell can comprise an antibody or fragment that recognizes a synthetic peptide (a peptide that does not occur in nature). For example, the antibody of the CAR can be an antibody that specifically recognizes a FLAG® tag or a fragment thereof. In some embodiments, the extracellular domain of the CAR can comprise an anti-HTP antibody or a fragment thereof.

[0052] When treating a human subject, the CAR of the administered CAR-T cells is preferably a fully human CAR or a humanized CAR to reduce immunogenicity to humans. In some embodiments, the extracellular domain of the CAR of the administered CAR-T cells comprises a humanized scFv that binds to the CAR-ID of the co-administered switch. The humanized scFv can comprise a humanized VH (variable heavy) sequence with non-human (e.g., murine) CDRs integrated into a human immunoglobulin framework. In some embodiments, the extracellular domain of the CAR can comprise a humanized anti-GCN4 scFv. Such scFv molecules can be derived from the anti-GCN4 scFv clone 52SR4 described in Zahnd et al., J. Biol. Chem. 279:18870-77, 2004. The humanized anti-GCN4 scFv (e.g., the humanized form of clone 52SR4) can comprise a humanized light chain, a humanized heavy chain, or a humanized light chain and a humanized heavy chain. Humanized anti-GCN4 (e.g., a humanized form of clone 52SR4) can comprise a humanized VH (variable heavy) sequence with non-human (e.g., murine) CDRs integrated into a human immunoglobulin framework. A specific example of such a humanized anti-GCN4 scFv molecule that can be used in the CAR-T cells of the invention includes the sequence shown in SEQ ID NO: 7, as in the sCAR-T cells exemplified in this disclosure.

[0053] The complementary and inactive CAR-T cells used in the present invention can be prepared according to methods known in the art or specific protocols exemplified in, for example, WO 2018 / 075807, WO 2015 / 057834, WO 2015 / 057852, or Marcu-Malina et al., Expert Opinion on Biological Therapy, Vol. 9, No. 5. In general, recombinant techniques can be used to introduce genetic material encoding a CAR into any suitable T cell, for example a human T cell, such as a central memory T cell. By way of example, the CAR-T cells used in the methods of the present invention can be generated by transduction of human T cells with a lentiviral vector expressing a genetically modified CAR (e.g. a humanized CAR). Several humanized CAR sequences and CAR-T cells harboring said humanized CAR sequences are known in the art. All of these can be easily used and / or adapted for use in the methods of the present invention. See, for example, WO 2018 / 075807, Li et al., Biomarker Research 8:36, 2020, and Maude et al., Blood 128:217, 2016.

[0054] As a specific example, the CAR-T switch used in the method of the present invention can comprise a humanized anti-CD19 antibody or an antigen-binding fragment thereof (e.g., Fab) fused to a GCN4 peptide. The humanized anti-CD19 antibody can comprise a light chain variable region sequence and a heavy chain variable region sequence identical or substantially identical (e.g., at least 95%, 96%, 97%, 98% or 99%) to SEQ ID NOs: 2 and 3, respectively. The GCN4 peptide CAR-ID can comprise an amino acid sequence identical or substantially identical (e.g., at least 95%, 96%, 97%, 98% or 99%) to SEQ ID NO: 1, and is fused to the anti-CD19 antibody at the N-terminus of the light chain. The complementary CAR-T cell used with the CAR-T switch comprises a humanized anti-GCN4 antibody or an antigen-binding fragment thereof (e.g., scFv) in its extracellular domain. The light chain variable region sequence and the heavy chain variable region sequence of the anti-GCN4 antibody are shown in SEQ ID NOs: 4 and 5, respectively. A specific anti-GCN4 scFv molecule with these two variable region sequences connected by a GS linker is shown in SEQ ID NO: 7. The CAR has a hinge region (SEQ ID NO: 8) that connects the C-terminus of the scFv to the N-terminus of the transmembrane domain (SEQ ID NO: 9). The intracellular domain of the CAR comprises the CD28 domain (SEQ ID NO: 10), the 4-1BB domain (SEQ ID NO: 11) and the CD3ζ domain (SEQ ID NO: 12) from the N-terminus to the C-terminus. The amino acid sequence of the entire CAR molecule is shown in SEQ ID NO: 6. The synthesis and production of the CD-19 targeting humanized CAR-T switch and complementary CAR-T cells are described in the art. See, for example, WO 2018 / 075807. Many other humanized CAR-T switches and complementary CAR-T cells suitable for use in the methods of the invention are also known. See, for example, WO 2018 / 075807 and Viaud et al., Proc. Natl. Acad Sci. USA 115:E10898-E10906, 2018.

[0055] SEQ ID NO:2 (humanized anti-CD19 light chain variable region with N-terminally fused GCN4 peptide) NYHLENEVARLKKLGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISKYLNWYQQKPGKAVKLLIYHTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGATLPYTFGQGTKLEIK SEQ ID NO:3 (humanized anti-CD19 heavy chain variable region) QVQLQESGPGLVKPSETLSVTCTVSGVSLPDYGVSWIRQPPGKGLEWLGVIWGSETTYYNSALKSRLTISKDNSKNQVSLKMSSLTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS SEQ ID NO:4 (humanized anti-GCN4 light chain variable region) QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYASWVQEKPDHLFRGLIGGTNNRAPGVPARFSGSLLGGKAALTISGAQPEDEAIYFCVLWYSDHWVFGGGTKLTVDG SEQ ID NO:5 (humanized anti-GCN4 heavy chain variable region) QVQLQQSGPGLVKPSETLSITCTTVSGFLLTDYGVNWVRQPPGKGLEWLGVIWGDGITDYNPSLKSRLTVSKDTSKNQVSLKMSSLTDADTARYYCVTGLFDYWGQGTTLTVSS SEQ ID NO:6 (Full sequence of humanized CAR) QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYASWVQEKPDHLFRGLIGGTNNRAPGVPARFSGSLLGGKAALTISGAQPEDEAIYFCVLWYSDHWVFGGGTKLTVDGGGGGSGGGG SGGGGSGGGGSQVQLQQSGPGLVKPSETLSITCTTVSGFLLTDYGVNWVRQPPGKGLEWLGVIWGDGITDYNPSLKSRLTVSKDTSKNQVSLKMSSLTDADTARYYCVTGLFDYWGQGTTL TVSSESKYGPPCPPCPDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPE EEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO:7 (humanized anti-GCN4 scFv) QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYASWVQEKPDHLFRGLIGGTNNRAPGVPARFSGSLLGGKAALTISGAQPEDEAIYFCVLWYSDHWVFGGGTKLTVDGGGGGSGGGGSG GGGSGGGGSQVQLQQSGPGLVKPSETLSITCTTVSGFLLTDYGVNWVRQPPGKGLEWLGVIWGDGITDYNPSLKSRLTVSKDTSKNQVSLKMSSLTDADTARYYCVTGLFDYWGQGTTLTVSS SEQ ID NO:8 (Hinge of humanized CAR) ESKYGPPCPPCPD SEQ ID NO:9 (Transmembrane domain of humanized CAR) FWVLVVVGGVLACYSLLVTVAFIIFWV SEQ ID NO: 10 (CD28 domain of the intracellular domain of humanized CAR) RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS SEQ ID NO: 11 (4-1 BB domain of the intracellular domain of humanized CAR) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL SEQ ID NO: 12 (CD3 ζ domain of the intracellular domain of humanized CAR) RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 15 (Full light chain sequence of CAR-T switch Fab SWI019) NYHLENEVARLKKLGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISKYLNWYQQKPGKAVKLLIYHTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGATLPYT FGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 16 (Full heavy chain sequence of CAR-T switch Fab SWI019) QVQLQESGPGLVKPSETLSVTCTVSGVSLPDYGVSWIRQPPGKGLEWLGVIWGSETTYYNSALKSRLTISKDNSKNQVSLKMSSLTAADTAVYYCAKHYYYGGSYAMDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC III. Human cancers to be treated The methods of the invention are directed to treating human cancer by directing sCAR-T cells to tumor cells in a human subject that carry one or more specific target molecules (e.g., CD19) that are recognized by the co-administered CAR-T switch. The switch may interact with multiple target cells expressing the same target molecule (e.g., CD19) or multiple target cells expressing different target molecules (e.g., CD19 and CD20). In some methods, the cancer cell surface molecule targeted by the sCAR-T platform of the invention is a receptor. The receptor may be an extracellular receptor. The receptor may be a cell surface receptor. By way of non-limiting example, the receptor may bind to a hormone, a neurotransmitter, a cytokine, a growth factor, or a cell recognition molecule. The receptor may be a transmembrane receptor. The receptor may be an enzyme-linked receptor. The receptor may be a G-protein couple receptor (GPCR). The receptor may be a growth factor receptor. The cell surface molecule may be a cell surface protein other than a receptor. The target molecule may be a differentiation protein group. By way of non-limiting example, the cell surface molecule may be selected from CD19, CD20, CD34, CD31, CD117, CD45, CD11b, CD15, CD24, CD114, CD182, CD14, CD11a, CD91, CD16, CD3, CD4, CD25, CD8, CD38, CD22, CD61, CD56, CD30, CD13, CLL1, CD33, CD123, or fragments or homologs thereof. In some preferred embodiments, the target molecule is CD19, as exemplified in the present disclosure.

[0056] A variety of human cancers can be treated with the methods of the invention. In some embodiments, the cancers treated are heterogeneous. In some embodiments, the cancers treated are malignancies of blood cells. For example, the cancers treated can originate from bone marrow cells or other blood cells. In these embodiments, the cancers can originate from B cells, T cells, monocytes, platelets, leukocytes, neutrophils, eosinophils, basophils, lymphocytes, hematopoietic stem cells, or endothelial cell precursors. In some embodiments, the cancers treated are relapsed, refractory B cell malignancies. In some preferred embodiments, the cancers treated originate from CD19 positive B lymphocytes. In some embodiments, the cancers can originate from stem cells. For example, the targeted cancer cells can originate from pluripotent cells. In some embodiments, the targeted cancer cells can originate from one or more endocrine glands. The endocrine glands can be lymphatic, pituitary, thyroid, parathyroid, pancreas, gonads, or pineal gland.

[0057] In some preferred embodiments, the methods of the invention are directed to treating subjects suffering from relapsed / refractory B-cell malignancies and who have been previously treated. Such diseases and conditions are well known in the art. See, for example, Swerdlow et al., WHO classification of tumours of haematopoietic and lymphoid tissues, World Health Organization Classification of Tumours, Lyon, France: IARC, Revised 4th Edition (2017), and Swerdlow et al., Blood 2016, 127(20):2375-90. In various embodiments, the subject can be a subject suffering from various forms of B-cell lymphoma, including unclassifiable, with characteristics intermediate between diffuse large B cell lymphoma (DLBCL) and classical Hodgkin's lymphoma.These include, for example, DLBCL (NOS), DLBCL associated with chronic inflammation, germinal center B cell type, activated B cell type, chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), transformation of follicular lymphoma (FL) and other low-grade indolent histology to DLBCL, CLL with Richter transformation, splenic marginal zone lymphoma, splenic B cell lymphoma / leukemia, unclassifiable (splenic diffuse red pulp small B cell lymphoma and hairy cell leukemia variant), extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), nodal marginal zone lymphoma, mantle cell lymphoma including in situ mantle cell neoplasia. lymphoma (MCL), follicular lymphoma (FL) (including germinal center confined follicular neoplasia and duodenal follicular lymphoma), large B-cell lymphoma with IRF4 rearrangement, primary cutaneous follicle center lymphoma, and Epstein Barr Virus (EBV)+ DLBCL (NOS) (including EBV+ DLBCL). Tumors suitable for treatment by the methods of the invention also include various primary mediastinal (thymic) large B-cell lymphomas. These include, for example, ALK+ large B-cell lymphoma, HHV8+ DLBCL(NOS), aggressive B-cell lymphomas with MYC and BCL2 and / or BCL6 rearrangements, aggressive B-cell lymphoma(NOS), as well as Burkitt's lymphoma, lymphoplasmacytic lymphoma (LPL) (including Waldenstrom macroglobulinemia (WM)).

[0058] Some methods of the invention are directed to the treatment of leukemia, such as CD-19 positive leukemia. Specific examples of leukemia include myeloid leukemia, lymphoblastic leukemia, myelogenous leukemia, acute myeloid leukemia, myelomonocytic leukemia, neutrophilic leukemia, myelodysplastic syndrome, B-cell lymphoma, Burkitt's lymphoma, large cell lymphoma, mixed cell lymphoma, follicular lymphoma, mantle cell lymphoma, Hodgkin's lymphoma, recurrent small lymphocytic lymphoma, hairy cell leukemia, multiple myeloma, basophilic leukemia, eosinophilic leukemia, megakaryoblastic leukemia, monoblastic leukemia, monocytic leukemia, erythroleukemia, erythroblastic leukemia, and hepatocellular carcinoma. In some embodiments, the disorder to be treated is a hematological malignancy. In some embodiments, the disorder to be treated is a B-cell malignancy. In some embodiments, the disorder being treated is chronic lymphocytic leukemia. In some embodiments, the disorder being treated is acute lymphocytic leukemia. In some embodiments, the disorder being treated is CD19-positive Burkitt's lymphoma.

[0059] In some preferred embodiments, the cancer to be treated is a CD19 positive tumor or malignancy. In some of these embodiments, the cancer to be treated is a B cell cancer or B cell malignancy. B cell cancer or B cell malignancy includes B cell lymphomas, which account for the majority of non-Hodgkin lymphomas (NHL). Examples of these B cell cancers include, for example, diffuse large B cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (Waldenstrom macroglobulinemia), hairy cell leukemia (HCL), primary central nervous system (CNS) lymphoma, and primary intraocular lymphoma.

[0060] IV. Administration and Dosage In carrying out the treatment of human cancer as described in this disclosure, a subject in need of treatment is administered a pharmaceutical composition comprising a therapeutically effective amount of the sCAR-T platform as described in this disclosure. As described in this disclosure, a therapeutically effective amount is of course also an amount that is optimal so as not to cause unacceptable toxicity in the subject to which the administration regimen is applied. In addition to the sCAR-T platform, the composition can include one or more pharma- ceutically acceptable carriers or agents, such as salts, excipients, or solvent agents. The pharma- ceutically acceptable carrier can be any suitable pharma- ceutically acceptable carrier. This can be one or more compatible solid or liquid fillers, diluents, other excipients, or encapsulating substances (e.g., physiologically acceptable carriers or pharmacologically acceptable carriers) suitable for administration to a human or veterinary patient. The administration of the sCAR-T platform can be in any order. For example, in some embodiments, the sCAR-T cells can be administered simultaneously with the administration of the CAR-T switch. In some other methods, the CAR-T switch can be administered prior to the administration of the sCAR-T cells. In some preferred embodiments, as exemplified in the present disclosure, sCAR-T cells are administered prior to administration of the CAR-T switch.

[0061] A general outline of the preparation and administration of the therapeutic compositions of the present invention is described in the art, see, for example, Goodman & Gilman's The Pharmacological Bases of Therapeutics, Hardman et al., eds., McGraw-Hill Professional (10th ed.). thP ed., 2001), Remington: The Science and Practice of Pharmacy, Gennaro, ed., Lippincott Williams & Wilkins (20P thP ed., 2003) and Pharmaceutical Dosage Forms and Drug Delivery Systems, Ansel et al. (eds.), Lippincott Williams & Wilkins (7P thPed., 1999). Methods for administering therapeutic compositions to subjects can be achieved based on routine procedures in the art. See, for example, Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 2000. th ed., 2000; Iwasaki et al., Jpn. J. Cancer Res. 88:861-6, 1997; Jespersen et al., Eur. Heart J. 11:269-74, 1990; and Martens, Resuscitation 27:177, 1994. Typically, compositions comprising sCAR-T platforms are administered in a physiologically tolerable medium, such as phosphate buffered saline (PBS) (e.g., administered by injection). Therapeutic compositions can be administered to subjects in need of treatment by any suitable route. In some preferred embodiments, the compositions are administered to subjects by parenteral administration. In these embodiments, the compositions administered naturally comprise a sterile aqueous formulation, and it is preferred that the sterile aqueous formulation is isotonic with the blood of the recipient.

[0062] A sufficient amount of CAR-T switch and complementary sCAR-T cells should be administered to the subject to inhibit tumor growth and promote tumor regression in the subject. A sufficient number of cells can be determined based on the type of tumor, the size and stage of development of the tumor, the route of administration, the age of the particular subject being treated, and other factors that will be readily apparent to one of ordinary skill in the art of tumor treatment. As a general guideline for administration by injection or implantation, approximately 1×10 cells per kg of body weight are administered. 5 ~Approx. 1×10 8 CARs + A single dose of sCAR-T cells comprising the cells is administered to the subject to be treated. Preferably, a single dose of sCAR-T cells is administered to the subject in a single administration, although it is reasonable to divide a single dose into multiple (e.g., 2, 3, or 4) administrations in the practice of the invention. In some embodiments, the dose is about 5×10 per kg of body weight. 6 ~Approx. 2×107 In various embodiments, the sCAR-T cell dose can include about 2.5×10 cells / kg body weight. 5 pieces, about 5×10 5 pieces, approximately 7.5×10 5 pieces, about 1×10 6 pieces, approximately 2.5×10 6 pieces, about 5×10 6 pieces, approximately 7.5×10 6 pieces, about 1×10 7 pieces, approximately 2.5×10 7 pieces, about 5×10 7 pieces, approximately 7.5×10 7 Alternatively, a subject of normal weight (e.g., around 70 kg, i.e., about 50 kg to about 90 kg) can be administered a fixed dose of sCAR-T cells. In various embodiments, the fixed CAR + T cell dose is approximately 20 x 10 cells 6 pieces, approximately 40×10 6 pieces, approximately 60×10 6 pieces, approximately 80×10 6 pieces, approximately 100×10 6 pieces, approximately 120×10 6 pieces, approximately 140×10 6 pieces, approximately 160×10 6 pieces, approximately 180×10 6 pieces, approximately 200×10 6 pieces, approximately 300×10 6 pieces, approximately 400×10 6 pieces, approximately 500×10 6 pieces, approximately 600×10 6 pieces, approximately 700×10 6 pieces, approximately 800×10 6 pieces, approximately 900×10 6 pieces, approximately 1000×10 6 The number can be 1 or more.

[0063] In some embodiments, the CAR + T cells approximately 35 x 10 6 ~Approx. 700×10 6 (or about 1400 x 10 6For a patient weighing 70 kg, this CAR + T cell dose is approximately 0.05 × 10 cells / kg body weight. 7 ~Approx. 1×10 7 (or 2 x 10 7 In some of these embodiments, as exemplified in the present disclosure, the amount of the cells is about 140×10 6 Approximately 700 x 10 cells 6 A fixed dose of 10 CAR T cells is administered to the subject. For a patient weighing 70 kg, this CAR T cell dose is approximately 0.2 × 10 cells per kg of body weight. 7 ~1×10 7 When treating subjects with significantly different body weights, the CAR-T cell dose on a per kg body weight basis can be adjusted accordingly. In some preferred embodiments, the dose is about 140×10 cells. 6 A fixed dose of 0.2×10 cells / kg body weight is administered to the subject, which is 0.2×10 cells / kg body weight for a subject weighing approximately 70 kg. 7 It is equivalent to one piece.

[0064] For CAR-T switch molecules, the daily dose can range from about 0.0001 mg to about 10 mg per kg of body weight. In some embodiments, the daily dose of the switch administered is from about 0.00025 mg to about 2.5 mg per kg of body weight. In some embodiments, the daily dose of the switch administered is anywhere from about 0.0005 mg (i.e., 0.00075 mg or 0.001 mg) to about 0.5 mg (i.e., 1 mg, 1.5 mg or 2 mg) per kg of body weight. In some embodiments, the daily dose of the switch administered is anywhere from about 0.01 mg (or 0.005 mg) to about 0.1 mg or about 1 mg per kg of body weight. In some embodiments, the daily dose of the switch administered is anywhere from about 0.045 mg to about 0.075 mg per kg of body weight. In various embodiments, the daily dose of CAR-T switch molecule administered can be about 0.0001 mg, about 0.00025 mg, about 0.0005 mg, about 0.00075 mg, about 0.001 mg, about 0.0025 mg, about 0.005 mg, about 0.0075 mg, about 0.01 mg, about 0.025 mg, about 0.05 mg, about 0.06 mg, about 0.075 mg, about 0.1 mg, about 0.25 mg, about 0.5 mg, about 0.75 mg, about 1 mg, about 1.5 mg, about 2 mg, about 2.5 mg, about 3 mg, about 4 mg, about 5 mg or more per kg of body weight. In some embodiments, the daily dose of the administered CAR-T switch molecule can be about 0.01 mg, about 0.025 mg, about 0.03 mg, about 0.04 mg, about 0.045 mg, about 0.05 mg, about 0.055 mg, about 0.06 mg, about 0.065 mg, about 0.070 mg, about 0.075 mg, about 0.085 mg, or about 0.095 mg. In some preferred embodiments, the daily dose of the administered CAR-T switch molecule is about 0.06 mg. As with the administration of CAR-T cells, the indicated doses (CAR-T switch molecules) can be administered in a single dose or multiple doses.

[0065] In some embodiments, the subject to be treated is administered CAR-T cells only at the beginning of treatment (e.g., one or two administrations), while the CAR-T switch molecule is subsequently administered multiple times during the treatment, e.g., the first few weeks or months, based on an "on / off schedule." As exemplified in the present disclosure, the on / off schedule comprises multiple cycles, each cycle including an "on" phase and an "off" phase. During the "on" phase, the CAR-T switch is administered to the subject, while during the "off" phase, the CAR-T switch is not administered. In various embodiments, during the "on" phase, which lasts for a certain period of time (e.g., any length of time from about one day to several weeks), the switch molecule can be administered to the subject twice a day, daily, every day, every third day, or more frequently. The "off" phase of the cycle can be any period of time longer than the interval between administrations of the "on" phase. Thus, for example, if administration occurs daily or every day during the "on" phase, the "off" phase can last for any length of time from about several days to about several years. In various embodiments, the "on" phase can last for, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks or more, and administration can be daily, every 2 days, every 3 days or more. For each of these "on" phases, the "off" phase can last for, for example, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year or more. The length of the period of the "on" phase and the "off" phase can be the same for each of the cycles. Alternatively, the duration of the "on" phase and / or the "off" phase can vary between different cycles. As a specific example, which is described in detail below, the treatment can include at least about 6 cycles. Each cycle lasts for 28 days, with the on and off phases being 7 and 21 days, respectively, as exemplified in the present disclosure.

[0066] The number of cells and frequency of administration will also vary depending on whether the treatment is for prophylactic or therapeutic purposes. For prophylactic purposes, a relatively small number of cells can be administered at relatively infrequent intervals over an extended period of time. Some subjects may continue to receive treatment for the rest of their lives. For therapeutic purposes, a relatively large number of cells may be required at relatively short intervals until tumor progression is reduced or eliminated, preferably until the subject's tumor is seen to partially or completely regress. The subject can then be administered a prophylactic regimen.

[0067] In some embodiments, the therapeutic compositions described herein can be administered locally by implanting a membrane, sponge, or other suitable material that has absorbed or encapsulated the sCAR-T platform disclosed herein into the affected area. When an implantation device is used, the device may be implanted into any suitable tissue or organ, and the CAR-T platform disclosed herein can be delivered directly through the device by bolus, directly through a continuous infusion, or directly through a catheter using continuous infusion.

[0068] As a specific example, some of the treatment methods of the present invention include administering approximately 0.35×10 CAR-T cells comprising the CAR sequence shown in SEQ ID NO:6 to a subject with a CD19-positive relapsed / refractory B-cell malignancy. 8 ~Approx. 7×10 8 The method involves administering (e.g., by infusion) a single dose of 1000 CAR-T cells, followed by administering to the subject one or more infusion cycles of a CAR-T switch molecule that is an anti-CD19 Fab antibody comprising the light and heavy chain variable region sequences shown in SEQ ID NOs: 2 and 3, respectively. Each infusion cycle includes (i) an "on" phase of about 5 to about 9 days, and (ii) an "off" phase of about 14 to about 28 days. During the on phase, a dose of about 0.045 mg to about 0.075 mg of switch per kg of body weight is administered daily. In some of these embodiments, the CAR-T cells used are autologous to the subject. In some embodiments, the infusion dose of CAR-T cells is about 1.4×10 cells. 8The daily dose of the CAR-T switch infused during the "on" phase is about 0.06 mg / kg body weight. In some embodiments, the "on" phase is about 7 days and the "off" phase is about 21 days. In various embodiments, the number of infusion cycles of administration of the CAR-T switch can be 2, 3, 4, 5, 6 or more. In some embodiments, prior to infusion of the CAR-T cells and the switch molecule, the subject is administered lymphodepleting preconditioning. In some of these embodiments, the lymphodepleting preconditioning is performed by chemotherapy using cyclophosphamide and fludarabine.

[0069] The sCAR-T platform described in this disclosure can be used in combination with other known regimens used to treat cancer. Methods for co-administration with other therapeutic agents are well known in the art. See, for example, Hardman, et al. (eds.) (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill, New York, NY; Poole and Peterson (eds.) (2001) Pharmacotherapeutics for Advanced Practice: A Practical Approach, Lippincott, Williams & Wilkins, Phila., Pa.; and Chabner and Longo (eds.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams & Wilkins, Phila., Pa.

[0070] example The following examples are provided to further illustrate the present invention, but are not intended to limit the scope of the invention. Other variations of the present invention will be readily apparent to those skilled in the art and are encompassed by the appended claims.

[0071] Example 1. IND-enabling studies of sCAR-T therapy for human cancer patients "Switchable" CAR-T (sCAR-T) therapy involves the activity of sCAR-T cells being controlled by an antibody-based switch. The switch targets a tumor antigen, and the sCAR recognizes a unique peptide embedded in the switch. The switch forms a bridge between the sCAR-T cell and the tumor cell, activating the sCAR-T cell and inducing tumor cell killing. The sCAR-T treatment platform used by the inventors includes a humanized inactive CAR-T cell (also referred to as CLBR001 in this disclosure) and a corresponding switch molecule (also referred to as SWI019 in this disclosure). The extracellular domain of the CAR of the CAR-T cell (SEQ ID NO:6) contains a humanized scFv antibody fragment (SEQ ID NO:7) that specifically binds to the GCN4 derivative peptide of the switch molecule (SEQ ID NO:1). The switch molecule further includes a CD19-targeting Fab antibody (having heavy and light chain variable region sequences shown in SEQ ID NOs:3 and 2, respectively), the light chain N-terminus of the antibody being fused to the GCN4 peptide. More detailed information on this sCAR-T platform is reported, for example, in WO 2018 / 075807. Although the switch molecule and sCAR-T cells are designed to have no activity on their own, their combination has been shown to completely eliminate tumors in xenograft and allograft models. The short half-life of the switch allows for rapid tuning of sCAR-T cell activity through switch administration. Furthermore, by swapping in different switches, a modular approach can be used to redirect sCAR-T cells to other tumor targets. Furthermore, cyclic on / off stimulation of sCAR-T cells has been shown to improve sCAR-T cell memory and persistence.

[0072] Using this exemplified sCAR-T platform, the inventors conducted IND-enabling studies to prepare and support first-in-human (FIH) clinical trials of sCAR-T therapy. Preclinical development of a platform that includes sCAR-T cells lacking endogenous antigen targets combined with an antibody-based switch molecule that lacks intrinsic activity in the absence of sCAR-T cells required the development of novel approaches. Since fidelity of such a system is essential for control, to ensure that CLBR001 cells are not activated in the presence of normal tissues, in vitro activity testing was performed by co-culturing CLBR001 cells with or without SWI019 and a panel of 14 primary cells, representing a landscape of major tissues throughout the body. Results showed that CLBR001 did not show activity in any of the 14 cell types tested, supporting the high fidelity of CLBR001 recognition of SWI019.

[0073] The lack of activity of SWI019 in the absence of CLBR001 cells did not qualify for traditional toxicology studies to identify a "no adverse effect level" (NOAEL) to support a first in human starting dose. In such cases, a minimal anticipated biological effect level (MABEL) is typically used to support a starting human dose based on predicted Cmax. However, the femtomolar in vitro activity of SWI019 in combination with CLBR001 resulted in a starting dose modeled to be well outside the range of possible clinical activity. Therefore, an in vivo approach to determine the MABEL was developed. In this study, NSG mice bearing CD19+Nalm-6 cell tumors were administered CLBR001 cells and single ascending doses of SWI019. Antitumor activity, peripheral cytokines, and the effective dose (ED) of SWI019 on CLBR001 cells in peripheral blood were evaluated. 50 Comparison of the IL-6 tumor burden and IL-6 tumor burden values ​​showed that the reduction in Nalm-6 tumor burden was the most sensitive marker of activity. Extravasation of CLBR001 cells from peripheral blood and weak ED of all three cytokines (IFN-γ, IL-2, and TNF-α) were not observed. 50 Therefore, antitumor activity (reduction in Nalm-6 tumor burden) was selected as the parameter used to determine the in vivo MABEL. Allometric scaling using mouse and NHP SWI019 PK data allowed the ED of the in vivo MABEL study to be determined. 20 "We modeled a recommended human dose of SWI019 that corresponds to a dose of 100 mg / kg / day ...

[0074] Figures 1 and 2 provide some more detailed information related to the following studies.

[0075] Figure 1 depicts an in vivo MABEL approach to assess FIH dose. In vitro MABEL simulations raised concerns about delivery of such small doses, potentially requiring multiple dosing cohorts of patients to reach a therapeutic dose. Therefore, an in vivo MABEL approach was developed using Nalm-6 xenografts. The study examined reduction in tumor burden, cytokines, and sCAR-T in peripheral blood.

[0076] Figure 2 summarizes the simulations of human equivalent doses of SWI019-switch, based on the most sensitive markers monitored in vitro and in vivo pharmacological analyses of the combined treatment of CLBR001 CAR-T cells and SWI019-switch.

[0077] In conclusion, the in vivo MABEL approach allowed us to determine a first-in-human (FIH) initiation switch dose (9.7 μg / kg) that is approximately 13,000-fold higher compared to the in vitro MABEL simulations, providing an excellent starting point for first-in-human trials that balances safety with potential patient benefit.

[0078] Example 2. Human testing of a switchable CAR-T cell platform for the treatment of tumors Based on the results of the above IND-enabling animal studies, we have initiated a human clinical trial (NCT04450069) of an on / off sCAR-T regimen (CLBR001+SWI019) for the treatment of relapsed / refractory B-cell malignancies. CD19-targeting chimeric antigen receptor (CAR) T cells are a transformative treatment option for patients with relapsed and refractory B-cell malignancies. Although significant responses have been seen in heavily pretreated patients, challenges remain with toxicities including cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), along with relapses related to tumor antigen loss. The sCAR-T treatment described in this disclosure is intended to address these challenges.

[0079] The phase 1, open-label, dose-escalation study was designed to evaluate the safety, tolerability, pharmacokinetics, pharmacodynamics, and clinical activity of the combination of CLBR001 and SWI019 in patients with relapsed / refractory B-cell malignancies. Autologous CLBR001 sCAR-T cells were produced from patient-derived apheresis material at a centralized manufacturing facility. After lymphodepletion with cyclophosphamide and fludarabine, patients received a single dose of CLBR001 cells followed by daily infusions of SWI019 for 7 days. The dose of CAR-T cells was approximately 140 × 10 cells. 6 From about 700 x 10 6 For a patient weighing 70 kg, a single dose of these CAR-T cells would be approximately 0.2 × 10 cells per kg of body weight. 7 ~1×10 7 The SWI019 switch molecule was administered in 28-day cycles for up to six cycles. Each cycle consisted of 7 days of daily IV administration followed by 21 days of no administration. Dose escalation of CLBR001 and SWI019 was determined by a [3+3] design in the initial cohorts and then by implementation of Bayesian Adaptive Design (BAYDE) decision rules.

[0080] Cohort 1 (CAR-T cells 140 × 10 6Three patients (2 with follicular lymphoma and 1 with mantle cell lymphoma) receiving CLBR001 + SWI019 switch 10 μg / kg) had evaluable safety and response data at the time of data cutoff. CLBR001 + SWI019 was well tolerated, with no DLTs observed in cohort 1. CLBR001 cell infusion was well tolerated, with no adverse events attributable to the cell product in any patient during the observation period prior to SWI019 administration, indicating that CLBR001 cells are inactive in the absence of SWI019. Increased serum cytokine concentrations and proliferation of CLBR001 in peripheral blood were observed only after administration of SWI019. Administration of SWI019 was well tolerated, with one case of simultaneous grade 1 CRS and grade 2 ICANS in cycle 2. The event subsided within 24 hours after administration of dexamethasone, and no recurrence of CRS was observed with continued administration of a reduced dose (50%) of SWI019, supporting the tunability of the platform. In the first cohort, two of three patients experienced a complete response by Lugano criteria.

[0081] This is the first report of a switchable CAR-T cell platform in patients with B cell malignancies. The combination of CLBR001+SWI019 was safe and well tolerated in patients with B cell malignancies, with promising clinical activity in cohort 1 using the lowest doses of both CLBR001 and SWI019.

[0082] Detailed information regarding the design and results of the human therapy is presented in Figures 3 and 4.

[0083] Figure 3 shows the efficacy of treatment in one patient as shown by CT scans pre-treatment, post-treatment cycle 3, and post-treatment cycle 6. Patient demographics, medical history, and treatment response are summarized at the top. The three CT scan images at the bottom show the target lesions in the mesenteric lymph nodes at the three time points, respectively. The results show that at baseline (pre-treatment), there were five target lesions: mesenteric, inguinal, iliac, and periaortic lymph nodes. The size of the mesenteric lymph node mass was 14.1 cm. The overall Deauville 5PS score by PET was 4. After treatment, all five FDG-avid target lesions were reduced. No new target masses were observed, and there is no evidence of lymphoma in the bone marrow by IHC. The patient continued to have a complete response (CR) by Lugano and RECIL 11 months after the start of treatment.

[0084] Figure 4 shows rapid resolution of toxicity in another treated patient. Panel A summarizes patient demographics, medical history, and treatment response. Panel B shows that discontinuing switch dosing on cycle 1 day 7 (C1D7) rapidly resolved fever despite continued CAR-T proliferation, demonstrating functional "off" switch capability. Panel C shows that SWI019 on cycle 2 day 1 administration rapidly resolved grade 1 cytokine release syndrome (Gr1 CRS) / grade 1 immune effector cell-associated neurotoxicity (Gr1 ICAN) with dexamethasone and continued dosing at 50% dose (5 μg / kg) did not induce further toxicity.

[0085] Results from human clinical trials indicate that the combination of CLBR001 sCAR-T cells + SWI019 switch molecule is safe and well tolerated. The combination shows promising signs of clinical activity in patients with existing B-cell malignancies. In summary, there were no adverse events related to CLBR001 prior to SWI019 administration, demonstrating the safety of CLBR001 cells alone and the accuracy of the CLBR001 + SWI019 interaction (i.e., CLBR001 does not act against endogenous human targets). The lowest dose of CLBR001 and SWI019 (cohort 1: 140 × 10 CAR-T cells) for maximum grade CRS / ICANS=1 was 6 Two CRs were achieved with CLBR001 at 10 μg / kg (100 mg / mL) and SWI019 at 10 μg / kg (100 mg / mL). Reactivation of CLBR001 with SWI019 from cycle 2 onwards demonstrated functional reversibility of the platform. Toxicity was rapidly resolved by maintaining or reducing the dose level of SWI019, indicating a potentially high safety profile using the CLBR001+SWI019 platform.

[0086] Example 3. Clinical trial to identify recommended phase II dose (RP2D) As shown in Figure 5, a dose-escalation phase I human clinical trial was conducted to investigate the optimal dosing regimen of CLBR001+SWI019 switchable CAR-T treatment for subjects with existing B-cell malignancies (CBR-sCAR19-3001, NCT04450069). Figure 5A outlines the design of the phase I human study. Eligibility criteria included relapsed / refractory B-cell malignancies including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL) and other histological findings. Patients with acute lymphoblastic leukemia (ALL), prior CAR-T cell therapy, and prior allogeneic stem cell transplant were excluded. The first two cohorts of the study tested dose escalation of SWITCH (SWI019) starting at 10 μg / kg and progressing to 30 μg / kg in a 3+3 dose escalation design. Subjects in the first two cohorts received 140e6 CAR+ cells prior to receiving SWI019. The optimal dose was determined in the next cohort using a Bayesian adaptive dose escalation (BAYDE) design. Cohort 4 evaluated SWI019 at 60 μg / kg with 140e6 CAR+ cells, and cohort 5 evaluated SWI019 at 30 μg / kg with 420e6 CAR+ cells. It may be possible to test the dose determined by the BAYDE model in cohort 5. The goal of the design was to validate the recommended Phase II CAR+ cell and SWI019 doses to be used in future studies. The demonstrated CLBR001 cell doses can be used with SWI019 in future studies and in combination with other switches.

[0087] As outlined in the treatment schedule shown in Figure 5B, subjects enrolled in the CBR-sCAR19-3001 study were treated as follows. Subjects were first asked to consent and then screened for eligibility. For subjects who met the predefined inclusion and exclusion criteria, leukoreduction products were shipped to a centralized manufacturing facility where T cells were isolated and transduced with sCAR vectors. After the cell products were manufactured and shipped, they were shipped back to the site where the subjects were treated. Subjects received lymphodepleting chemotherapy (usually cyclophosphamide and fludarabine) before being infused with CLBR001 cells. Subjects were then treated with a 28-day cycle of SWI019, with 7 days of daily administration followed by 21 days of no administration. Subjects were evaluated for dose-limiting toxicity, response (with PET / CT every 28 days as needed), and pharmacodynamic biomarkers (cytokines in peripheral blood), among others. [Table 1] Of the first nine subjects with data available at the time of data cutoff, seven had a partial response (PR) or complete response (CR) (78%) and six had a CR (67%). Two of the nine subjects had grade 3 or higher cytokine release syndrome (CRS), and none of the subjects had grade 3 or higher immune effector cell-associated neurotoxicity (ICANS). Among the first nine subjects, the median time to resolution of any grade of CRS was 1 day, and the median time to resolution of any grade of ICANS was 3 days. This compares favorably with the median time to resolution of CRS or ICANS for the three FDA-approved CAR-T cell products. The reduction in duration of CRS is expected to be due to the ability to withhold or reduce dosing of SWI019, which is not possible with previous CAR-T cell products.

[0088] Results of the first 14 subjects in the first four cohorts treated in the CBR-sCAR19-3001 study are shown in Table 2. No dose-limiting toxicity (DLT) occurred in the first three cohorts, and two DLTs occurred in cohort 4. DLTs were defined as treatment-related adverse events occurring during the DLT period, which occurred 35 days after the first dose of SWI019 in cohorts 1 and 2, and 28 days after the first dose of SWI019 in cohorts 3 and 4. Late DLT responses were defined as treatment-related adverse events that met the DLT criteria and occurred after the DLT window. Efficacy responses were defined as subjects who experienced a PR or CR. At the time of data cutoff, the results were pending in two subjects and unknown in one subject. Pharmacodynamic response (PD) was defined as a subject with a 3-fold increase in peripheral blood cytokine concentrations after SWI019 administration. [Table 2] To determine the optimal switch and dose (OSD) of CLBR001, the BAYDE model was employed using the data in the above table (Figure 6). BAYDE is a dose-finding design that uses dose-toxicity / response curves based on historical and cumulative study data to recommend a safe, effective and efficacious dosing regimen using Bayesian adaptive modeling and a Bayesian logistic regression model (BRLM). The recommended dose is optimally selected across the dose range. The BAYDE approach supports better decision-making using an initial dose relationship based on historical safety, toxicity, PD and efficacy data, a better constructed Bayesian modeling approach and an updated dose relationship during the study. The goal of dose selection is (a) to provide optimal dose recommendations using available data, (b) to identify a maximum tolerated dose (MTD) or (c) to identify an OSD level below the MTD that shows an acceptable PD and / or efficacy response. Dose levels are (a) safe if the risk of DLT rate >33% is <25% (safety tolerance threshold) and (b) demonstrate acceptable PD and / or efficacy response if the estimated response rate is at least 50%. The BAYDE model for this study used four endpoints for dose selection: 1: binary variable for standard DLT, 2: binary variable for delayed DLT, 3: binary variable for efficacy response, and 4: binary variable for PD response. Acceptable dose ranges were 10-1000μg / kg for SWI019 and 140-700e6 CAR+ cells for CLBR001.

[0089] To calculate a clinical utility index for ranking combinations of CLBR001 and SWI019 treatments, the model was fed with cumulative subject data, the response model was updated, and posterior probabilities were estimated using Bayesian methods and simulations. The clinical utility index shown in Figure 6 is optimal at 100, which represents a good balance between efficacy and potential toxicity. The above clinical utility index assumes that efficacy of withheld subjects is "yes" and efficacy of unknown subjects is "no". Thus, the optimal switch and CLBR001 doses selected for expansion were 60μg / kg SWI019 and 140e6 CAR+ cells.

[0090] Although the foregoing invention has been described in some detail by way of illustration and example for ease of understanding, it will be readily apparent to those skilled in the art, in light of the teachings of the present invention, that certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

[0091] All publications, databases, GenBank sequences, patents, and patent applications cited in this specification are incorporated by reference into this disclosure as if each was specifically and individually indicated to be incorporated by reference.

Claims

1. A pharmaceutical combination for use in a method for treating a CD19-positive malignant tumor in a human subject, inhibiting the growth of said tumor, and / or promoting the regression of said tumor, the method comprising the steps of administering to the subject: (a) a pharmaceutical comprising a chimeric antigen receptor T-cell switch molecule (CAR-T switch) comprising an anti-CD19 Fab antibody having the light chain variable region and heavy chain variable region sequences shown as SEQ ID NOs: 2 and 3, respectively; and (b) a pharmaceutical comprising a complementary CAR-T cell comprising the CAR sequence shown in SEQ ID NO: 6, thereby treating a B-cell malignant tumor in said subject, inhibiting the growth of said tumor, and / or promoting the regression of said tumor.

2. The pharmaceutical combination of claim 1, wherein the anti-CD19 Fab antibody comprises the light and heavy chain sequences shown as SEQ ID NOs: 15 and 16, respectively.

3. 2. The pharmaceutical combination of claim 1, wherein the subject is administered a single dose of the CAR-T cells at the start of treatment and multiple doses of the CAR-T switch over the course of the treatment.

4. 4. The pharmaceutical combination of claim 3, wherein the subject is infused with a single dose of the CAR-T cells, followed by one or more infusion cycles of the CAR-T switch, each cycle comprising an "on" phase during which the CAR-T switch is infused daily for about 5 to about 9 days, and an "off" phase during which no CAR-T is administered for about 14 to about 28 days.

5. The dose of the CAR-T cells administered to the subject is about 60 x 10 cells ６ pieces, approximately 80 x 10 ６ pieces, approximately 100 x 10 ６ pieces, approximately 120 x 10 ６ pieces, approximately 140 x 10 ６ pieces, approximately 160 x 10 ６ pieces, approximately 180 x 10 ６ pieces, approximately 200 x 10 ６ pieces, approximately 300 x 10 ６ pieces, approximately 400 x 10 ６ pieces, approximately 500 x 10 ６ pieces, approximately 600 x 10 ６ pieces, approximately 700 x 10 ６ pieces, approximately 800 x 10 ６ pieces, approximately 900 x 10 ６ pieces, approximately 1000 x 10 ６ 4. The pharmaceutical combination of claim 3, wherein the number of the active ingredient is 1 or more.

6. The dose of the CAR-T cells administered to the subject is about 0.35 x 10 cells ８ From about 14 x 10 ８ The pharmaceutical combination according to claim 3, wherein the amount is 100 mg / kg or more.

7. The dose of the CAR-T cells administered to the subject is about 1.4 x 10 cells ８ From about 7 x 10 ８ The pharmaceutical combination according to claim 3, wherein the amount is 100 mg / kg or more.

8. The dose of the CAR-T cells administered to the subject is about 1.4 x 10 cells ８ The pharmaceutical combination according to claim 3, wherein the amount is 100 mg / kg or more.

9. The combination pharmaceutical of claim 3, wherein the dose of the CAR-T switch administered to the subject is about 0.01 mg to about 0.1 mg per kg of body weight.

10. The pharmaceutical combination of claim 9, wherein the dose of the CAR-T switch administered to the subject is about 0.01 mg, about 0.025 mg, about 0.03 mg, about 0.04 mg, about 0.045 mg, about 0.05 mg, about 0.055 mg, about 0.06 mg, about 0.065 mg, about 0.070 mg, about 0.075 mg, about 0.085 mg, or 0.095 mg per kg of body weight.

11. The pharmaceutical combination of claim 9, wherein the dose of the CAR-T switch administered to the subject is from about 0.045 mg / kg to about 0.075 mg / kg of body weight.

12. The pharmaceutical combination of claim 9, wherein the dose of the CAR-T switch administered to the subject is about 0.06 mg / kg of body weight.

13. The combination drug of claim 1, wherein the CD19-positive malignant tumor is a CD19-positive B-cell cancer.

14. 2. The pharmaceutical combination of claim 1, wherein the CD19-positive malignancy is a relapsed / refractory B-cell malignancy.

15. 1. A pharmaceutical combination for use in a method of treating a CD19-positive relapsed / refractory B-cell malignancy in a human subject, inhibiting the growth of said tumor and / or promoting the regression of said tumor, said method comprising: (a) administering to a human subject about 0.35 x 10 CAR-T cells; ８ pieces ~ approx. 7×10 ８ (b) administering to the subject a single dose of a medicament comprising a CAR-T switch molecule during one or more cycles, each cycle comprising (i) an "on" phase of about 5 to about 9 days and (ii) an "off" phase of about 14 to about 28 days, wherein a daily dose of about 0.045 mg to about 0.075 mg per kg of body weight is administered during the "on" phase, and the CAR-T switch molecule comprises an anti-CD19 Fab antibody comprising light chain and heavy chain sequences set forth as SEQ ID NOs: 15 and 16, respectively, thereby treating the CD19-positive relapsed / refractory B-cell malignancy in the subject, inhibiting growth of the tumor, and / or promoting regression of the tumor.

16. The pharmaceutical combination of claim 15, wherein the CAR-T cells are autologous cells of the subject.

17. The injection dose of the CAR-T cells was approximately 1.4 x 10 cells ８ 16. The pharmaceutical combination of claim 15, wherein the daily dose of the CAR-T switch infused during the "on" phase is about 0.06 mg / kg body weight.

18. 16. The pharmaceutical combination of claim 15, wherein the "on" phase is about 7 days and the "off" phase is about 21 days.

19. 16. The pharmaceutical combination of claim 15, wherein the number of cycles of CAR-T switch infusion is 2, 3, 4, 5, 6, 7 or more.

20. The combination pharmaceutical of claim 15, wherein the subject undergoes lymphodepleting preconditioning before the CAR-T cells and the switch molecule are administered.

21. 21. The pharmaceutical combination of claim 20, wherein the lymphodepleting preconditioning is chemotherapy with cyclophosphamide and fludarabine.

22. 16. The combination pharmaceutical of claim 15, wherein the CD19-positive relapsed / refractory B-cell malignancy is selected from the group consisting of diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), hairy cell leukemia (HCL), primary intraocular lymphoma, Burkitt's lymphoma, and Waldenstrom's macroglobulinemia.

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